Compositions and methods for treating and preventing inflammation

ABSTRACT

The present invention provides novel compounds compositions and methods for (i) treating or preventing inflammation; and (ii) preventing or reducing hyperactivation of innate immune response, by inhibiting NRP1-dependent cell-signaling. Also provided are compounds, composition, and methods of specifically inhibiting SEMA3A-mediated cell signaling.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. provisional application Ser. No. 62/046,459, filed on Sep. 5, 2014, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form entitled “12810_513_ST25”, created on Sep. 8, 2015 having a size of 580 Kbytes. The computer readable form is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N.A.

FIELD OF THE INVENTION

The present invention relates to inflammation. More specifically, the present invention is concerned with the inhibition of the NRP1 pathway for the prevention or treatment of inflammation.

REFERENCE TO SEQUENCE LISTING

N.A.

BACKGROUND OF THE INVENTION

Local acute inflammatory responses are predominantly beneficial and constitute the body's first line of defense against infection of the host. Conversely, acute systemic inflammation such as in septic shock is a leading cause of morbidity and mortality (58). When chronic, low-grade inflammation persists, it can be at the origin of a several systemic diseases ranging from type II Diabetes Mellitus, arthritis, cancer, a number of neuro-inflammatory conditions and more.

Of all cytokines, receptors and other players thought to contribute to the inflammatory processes, one paradigm that has been largely overlooked is the influence of classical neuronal guidance cues and their receptors. These include semaphorin3A (SEMA3A, e.g., mRNA: NM_006080; and protein: NP_006071 and FIG. 21) and their receptor Neuropilin-1 (NRP1, e.g., mRNA: NM_001024628; and protein: NP_001019799, NM_003873 and FIG. 22 (isoform 2 or b, secreted) and 26 (isoform 1). NRP1 is expressed on both lymphoid and myeloid cells (59, 31). Yet its role in inflammation is largely unknown and especially in the context of cytokine production.

The Semaphorins were initially characterized as key players in axonal guidance during embryogenesis. It is now clear that the role of Semaphorins extends beyond axonal guidance and influence vascular systems, tumor growth and the immune response. The Semaphorin family counts at least 21 vertebrate genes and 8 additional genes in invertebrates. All Semaphorins contain a ˜500 amino acid SEMA domain that is required for signaling. Class 3 Semaphorins (such as SEMA3A) are the only secreted members of the family. SEMA3A is synthesized as a disulphide-linked homodimer and dimerization is essential for signaling.

In neurons, binding of SEMA3A to its cognate receptor Neuropilin-1 (NRP1) provokes cytoskeletal collapse via plexins (60); the transduction mechanism in endothelial cells remains ill-defined. NRP1 has the particular ability to bind two structurally dissimilar ligands via distinct sites on its extracellular domain (27-29). It binds not only SEMA3A (46, 47) provoking cytoskeletal collapse but also VEGF₁₆₅ (28, 29, 47, 61) enhancing binding to VEGFR2 and thus increasing its angiogenic potential (62). Crystallographic evidence revealed that VEGF₁₆₅ and SEMA3A do not directly compete for NRP1 but rather can simultaneously bind to NRP1 at distinct, non-overlapping sites (63). Moreover, genetic studies show that NRP1 distinctly regulates the effects of VEGF and SEMA3A on neuronal and vascular development (64). Finally, NRP1 has also been found to bind to TGF-β1 and to regulate its latent form.

NRP1 is a single-pass transmembrane receptor with a large 860 amino acid extracellular domain subdivided into 3 sub-domains (a1a2, b1b2 and c) and a short 40 amino acid intracellular domain (65). In neurons, binding of SEMA3A to NRP1 recruits Plexins, which transduce their intracellular signal (60) and provoke cytoskeletal collapse. The transduction mechanism in endothelial cells remains ill-defined. NRP1 binds SEMA3A (46, 47) primarily via its a1a2 (but possibly also b1-) domain (provoking cytoskeletal collapse) and VEGF₁₆₅ (28, 29, 47, 61) via its b1b2 domain (enhancing binding to VEGFR2 and thus increasing its angiogenic potential (62). The elevated levels of SEMA3A in the ischemic retina may thus partake in forcing neovessels into the vitreous by collapsing and deviating the advancing tip cells away from the source of the repellent cue (21).

The CNS had long been considered an immune-privileged system, yet it is now clear that the brain, retina and spinal cord are subjected to complex immune-surveillance (1, 2). Immunological activity in the CNS is largely dependent on an innate immune response and is present in health and heightened in diseases such as diabetic retinopathy, multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer's disease. This is apparent in the retina where an intensified, largely microglial/macrophage-based immune response is associated with the progression of several sight threatening diseases such as diabetic retinopathy (DR)(3-5), age related macular degeneration (AMD)(6-8) and retinopathy of prematurity (ROP)(9, 10). Together, these retinal diseases account for the principal causes of loss of sight in industrialized countries (6, 11, 12).

Many of the current line of treatments of inflammatory diseases and conditions suffer from important side-effects and deficient long-term safety profiles. Accordingly, there remains a need for novel pharmaceutical targets and methods of treatments.

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present inventors have sought to determine the function of myeloid-resident NRP1 in the context of the innate immune response.

The present inventors have determined that SEMA3A, VEGF and TGF-3 act as potent attractants for mononuclear phagocytes (MPs, e.g., microglia and macrophages) expressing the NRP1 receptor. Inhibition of NRP1 signaling in innate immune cells was shown to result in protection against MPs-dependent inflammation and tissue damage under a variety of conditions involving hyperactivation of the innate immune-response including proliferative retinopathies, septic shock and cerebral ischemia/stroke. Furthermore, the present inventors have designed various soluble NRP1-derived traps which inhibit SEMA3A signalling and shown that inhibition of SEMA3A significantly reduce the inflammatory response in various conditions.

Accordingly, the present invention relates to the inhibition of NRP1 cell signalling (e.g., NRP1 and its ligands) for the prevention or treatment of inflammatory diseases and conditions involving hyperactivation (i.e., pathological activation) of the innate immune response. Non-limiting examples of such disease and conditions include sepsis, stroke, cerebral ischemia, and various proliferative retinopathies.

More specifically, in an aspect, the present invention concerns a method of treating or preventing inflammation comprising inhibiting NRP1-dependent cell-signaling.

In another aspect, the present invention relates to a method of preventing or reducing hyperactivation of innate immune response comprising inhibiting NRP1-dependent cell-signaling. In an embodiment, the hyperactivation of innate immune response comprises i) secretion of IL-1β and TNFα and/or activation/recruitment of mononuclear phagocytes (MPs).

In an embodiment, inhibiting NRP1-dependent cell-signaling comprises: a) reducing NRP1 expression or activity; and/or b) reducing NRP1 ligand expression or activity. In an embodiment, the NRP1 ligand is SEMA3A, VEGF₁₆₅ or TGF-β. In a particular embodiment, the NRP1 ligand is SEMA3A.

In an embodiment, reducing NRP1 activity consists of inhibiting the binding of NRP1 to at least one NRP1 ligand. In an embodiment, inhibiting the binding of NRP1 to at least one NRP1 ligand comprises administering an NRP1 antibody (e.g., a SEMA3A antibody).

In another embodiment of the above methods, reducing NRP1 activity comprises administering an effective amount of an NRP1 trap which comprises soluble NRP1 polypeptide or a functional fragment thereof. In a particular embodiment, the NRP1 trap is as set forth in FIG. 19 or 20.

In a particular embodiment, the NRP1 trap of the present invention inhibits the binding of SEMA3A to NPR-1 but does not substantially inhibit the binding of VEGF to NRP1. In an embodiment, such NRP1 trap comprises the a1a2 domain of NRP1 but does not comprise the b1 and/or b2 subdomain(s) of NRP1. In another embodiment, such trap comprises a mutation in domain b1 at a position corresponding to tyrosine 297 of the NRP1 amino acid sequence as set forth in FIG. 22 which reduces or abrogates VEGF binding to the trap. In a specific embodiment, the mutation changes the tyrosine at position 297 to an alanine.

In specific embodiments, the NRP1 trap of the present invention: a) comprises domains a1, a2, b1, b2 and c and of NRP1; b) comprises domains a1, a2, b1 and b2 of NRP1; c) comprises domains a1, a2 and b1 of NRP1; d) comprises domains a1 and a2 of NRP1; e) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which reduces or abrogates the binding to VEGF; f) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which changes the tyrosine to an alanine; g) does not comprise domain c of NRP1; h) does not comprise domain b1 of NRP1; i) does not comprise domains b1 and b2 of NRP1; or j) does not comprise domains b1, b2 and c of NRP1.

In an embodiment of the above methods, inhibiting NRP1 ligand expression or activity comprises specifically inhibiting SEMA3A expression or SEMA3A binding to NRP1. In a particular embodiment, inhibiting SEMA3A binding to NRP1 comprises administering a SEMA3A antibody.

In a particular embodiment, the method of the present invention comprises reducing NRP1 expression by administering a NRP1 antisense, shRNA or siRNA.

In another embodiment, the method comprises reducing SEMA3A expression by administering a SEMA3A antisense, shRNA or siRNA.

In a further aspect, the present invention concerns a compound for the prevention or treatment of inflammation wherein the compound a) reduces NRP1 expression or activity; or b) reduces NRP1 ligand expression or activity.

In another aspect, the present invention relates to a compound for preventing or reducing hyperactivation of innate immune response, wherein the compound a) reduces NRP1 expression or activity; or b) reduces NRP1 ligand expression or activity.

In an embodiment, the compound is: i) A SEMA3A antibody; ii) A NRP1 antibody; iii) A NRP1 trap; iv) A SEMA3A antisense, shRNA or siRNA; or v) A NRP1 antisense, shRNA or siRNA. In another particular embodiment the compound is a NRP1 antibody or a NRP1 trap and said compound does not substantially reduce the binding of VEGF to NRP1.

In a particular embodiment, the compound is a NRP1 trap. In an embodiment, the NRP1 trap is as set forth in FIGS. 19, 20, 27 and Table 1.

In another embodiment, the NRP1 trap of the present invention inhibits the binding of SEMA3A to NPR-1 but does not substantially inhibit the binding of VEGF to NRP1. In an embodiment, such NRP1 trap comprises the a1a2 domain of NRP1 but does not comprise the b1 and/or b2 subdomain(s) of NRP1. In another embodiment, such trap comprises a mutation in domain b1 at a position corresponding to tyrosine 297 of the NRP1 amino acid sequence as set forth in FIG. 22 which reduces or abrogates VEGF binding to the trap. In a specific embodiment, the mutation changes the tyrosine at position 297 to an alanine.

In specific embodiments, the NRP1 trap of the present invention: a) comprises domains a1, a2, b1, b2 and c and of NRP1; b) comprises domains a1, a2, b1 and b2 of NRP1; c) comprises domains a1, a2 and b1 of NRP1; d) comprises domains a1 and a2 of NRP1; e) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which reduces or abrogates the binding to VEGF; f) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which changes the tyrosine to an alanine; g) does not comprise domain c of NRP1; h) does not comprise domain b1 of NRP1; i) does not comprise domains b1 and b2 of NRP1; or j) does not comprise domains b1, b2 and c of NRP1.

In an embodiment, the NRP1 trap of the present invention comprises: (i) amino acids 1-856 (preferably, 22 to 856) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (ii) amino acids 1 to 583 (preferably 22 to 583) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (iii) amino acids 1 to 424 (preferably 22-424) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (iv) amino acids 1 to 265 (preferably 22 to 265) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (v) 1 to 430 and 584 to 856 (preferably 22-430 and 584-856) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vi) amino acids 1 to 274 and 584 to 856 (preferably 22-274 and 584 to 856) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vii) amino acids 1 to 430 and 584 (preferably 22 to 430 and 584 to 856) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69). In a particular embodiment, the above noted traps comprise one or more mutation to reduce VEGF or SEMA3A binding as described above.

In another aspect, the present invention provides compositions for i) treating and preventing inflammation or ii) for preventing or reducing the hyperactivation of the innate immune response, comprising one or more compounds of the present invention together with a pharmaceutical carrier.

The present invention also relates to the use of one or more compounds of the present invention in the manufacture of a medicament for i) treating and preventing inflammation or ii) for preventing or reducing the hyperactivation of the innate immune response.

In a related aspect, the present invention concerns the use of one or more compounds of the present invention for i) treating and preventing inflammation or ii) for preventing or reducing the hyperactivation of the innate immune response.

In a particular embodiment, the methods, compounds (e.g., NRP1 polypeptide traps, nucleic acids encoding same, vectors, cells comprising vectors, etc.), compositions and uses of the present invention are for treating or preventing inflammatory diseases and conditions selected from the group consisting of septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis, diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease or stroke.

In an embodiment, the methods, compounds, compositions and uses of the present invention are for treating or preventing septic shock, cerebral ischemia or stroke.

More specifically, in accordance with the present invention, there is provided the following items:

1. A method of treating or preventing inflammation comprising inhibiting NRP1-dependent cell-signaling in a subject.

2. A method of preventing or reducing hyperactivation of innate immune response comprising inhibiting NRP1-dependent cell-signaling in a subject.

3. The method of item 2, wherein said hyperactivation of innate immune response comprises i) secretion of IL-6, IL-1β and TNFα and/or recruitment of mononuclear phagocytes (MPs).

4. The method of any one of items 1-3, wherein inhibiting NRP1-dependent cell-signaling comprises: (a) reducing NRP1 expression or activity; and/or (b) reducing NRP1 ligand expression or activity; wherein said NRP1 ligand is SEMA3A, VEGF and/or TGF-β.

5. The method of item 4, wherein the method comprises (i) reducing NRP1 activity by inhibiting the binding of NRP1 to at least one NRP1 ligand.

6. The method of item 5, wherein the NRP1 ligand is SEMA3A, VEGF or TGF-β.

7. The method of item 5 or 6, wherein inhibiting the binding of NRP1 to at least one NRP1 ligand comprises administering an anti-NRP1 antibody or an NRP1 trap, wherein said trap comprises a NRP1 polypeptide or a functional fragment or variant thereof.

8. The method of item 7, wherein said NRP1 polypeptide corresponds to soluble NRP1 isoform 2.

9. The method of item 8, wherein said soluble NPR1 isoform 2 comprises or consists essentially of a polypeptide having an amino acid sequence as set forth in FIG. 22 without a signal peptide.

10. The method of item 7, wherein said NRP1 polypeptide corresponds to the extracellular domain of an NRP1 isoform 1 polypeptide.

11. The method of item 10, wherein said NRP1 isoform 1 polypeptide is as set forth in FIG. 26 and wherein said extracellular domain comprises amino acids 22 to 859 corresponding to the NRP1 polypeptide shown in FIG. 26 (SEQ ID NO:66).

12. The method of any one of items 7 to 11, wherein said NRP1 trap comprises an NRP1 polypeptide comprising (i) amino acids 22 to 609 of a NRP1 polypeptide as set forth in SEQ ID NO: 65; (ii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 66; (iii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 69 (iv) or a functional fragment or functional variant of (i), (ii) or (iii).

13. The method of any one of items 7 to 12, wherein said anti-NRP1 antibody inhibits the binding of SEMA3A to NPR-1 but does not substantially inhibit the binding of VEGF to NRP1 and wherein said NRP1 trap binds to SEMA3A but does not substantially bind to VEGF165 or has a reduced binding affinity for VEGF165 compared to SEMA3A binding affinity.

14. The method of item 13, wherein said NRP1 trap (i) lacks completely or partially domain b1 and/or b2 of NRP1; or (ii) comprises at least one amino acid point mutation which inhibits VEGF binding to NRP1.

15. The method of item 13, wherein said anti-NRP1 antibody does not bind to domain b1 and/or b2 of NRP1.

16. The method of item 14, wherein said point mutation comprises (a) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to tyrosine 297 of an NRP1 amino acid sequence set forth in FIG. 22 or FIG. 26; (b) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to aspartic acid 320 of an NRP1 amino acid sequence set forth in FIG. 22 or FIG. 26; and/or (c) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to glutamic acid 319 of an NRP1 amino acid sequence set forth in FIG. 22 or FIG. 26.

17. The method of item 16, wherein said point mutation is a Y297A substitution; a D320K substitution and/or a E319K substitution.

18. The method of any one of item 7 to 12, wherein said NRP1 trap: (a) comprises domains a1, a2, b1, b2, and c and of said NRP1 polypeptide; (b) comprises domains a1, a2, b1 and b2 of said NRP1 polypeptide; (c) comprises domains a1, a2, and b1 of said NRP1 polypeptide; (d) comprises domains a1 and a2 said NRP1 polypeptide; (f) comprises domain b1 of said NRP1 polypeptide, wherein said domain b1 comprises at least one point mutation at an amino acid residue corresponding to (i) tyrosine 297; (ii) aspartic acid 320 and/or (iii) glutamic acid 319, of a NRP1 polypeptide comprising an amino acid sequence as set forth in FIG. 26, wherein said at least one mutation reduces or abrogates binding to VEGF₁₆₅; (g) lacks completely or partially domain c of said NRP1 polypeptide; (h) lacks completely or partially domain b1 of said NRP1 polypeptide; (i) lacks completely or partially domain b2 of said NRP1 polypeptide; (j) lacks domains b1 and b2 of said NRP1 polypeptide; or (k) lacks domains b1, b2 and c of said NRP1 polypeptide.

19. The method of item 18, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 27 to 141 of an NRP1 polypeptide as set forth in FIG. 26; (ii) said domain a2 comprises an amino acid sequence corresponding to amino acids 147 to 265 of an NRP1 polypeptide as set forth in FIG. 26; (iii) said domain b1 comprises an amino acids sequence corresponding to amino acids 275 to 424 of an NRP1 polypeptide as set forth in FIG. 26; (iv) said domain b2 comprises an amino acids sequence corresponding to amino acids 431 to 583 of an NRP1 polypeptide as set forth in FIG. 26; and/or (v) said domain c domain comprises an amino acids sequence corresponding to amino acids 645 to 811 of an NRP1 polypeptide as set forth in FIG. 26.

20. The method of item 18, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 22 to 148 of an NRP1 polypeptide as set forth in FIG. 26; (ii) said domain a2 comprises an amino acid sequence corresponding to amino acids 149 to 275 of an NRP1 polypeptide as set forth in FIG. 26; (iii) said domain b1 comprises an amino acids sequence corresponding to amino acids 276 to 428 of an NRP1 polypeptide as set forth in FIG. 26; (iv) said domain b2 comprises an amino acids sequence corresponding to amino acids 429 to 589 of an NRP1 polypeptide as set forth in FIG. 26; and/or (v) said domain c domain comprises an amino acids sequence corresponding to amino acids 590 to 859 of an NRP1 polypeptide as set forth in FIG. 26.

21. The method of item 7, wherein said method comprises inhibiting the binding of NRP1 to at least one NRP1 ligand by administering a NRP1 trap consisting essentially of a trap as set forth in Table 1 or a functional variant thereof.

22. The method of any one of items 7 to 20, wherein said NRP1 trap further comprises a protein purification domain.

23. The method of 22, wherein said purification domain is a polyhistidine tag.

24. The method of any one of items 7 to 20, wherein said NRP1 trap further comprises a FC domain.

25. The method of any one of items 22 to 24, wherein said NRP1 trap comprises a protease or peptidase cleavage site enabling said protein purification domain or FC domain to be removed from said NRP1 trap.

26. The method of item 25, wherein said protease or peptidase is a TEV protease cleavage site.

27. The method of item 26, wherein said TEV protease cleavage site comprises the amino acid sequence GSKENLYFQG.

28. The method of item 4, wherein the method comprises reducing NRP1 ligand expression or activity, and wherein the NRP1 ligand is SEMA3A.

29. The method of item 28, comprising reducing SEMA3A activity by inhibiting SEMA3A binding to NRP1 by administering an anti-SEMA3A antibody which binds to the SEMA domain of SEMA3A [ajouter support pour définir domaine spécifique].

30. The method of 4, wherein said method comprises reducing NRP1 expression by administering a NRP1 antisense, shRNA or siRNA.

31. The method of 4, wherein said method comprises reducing SEMA3A expression by administering a SEMA3A antisense, shRNA or siRNA.

32. A NRP1 polypeptide trap comprising s a NRP1 polypeptide or a functional fragment or variant thereof which binds to SEMA3A, VEGF165 and/or TGF-β.

33. The NRP1 polypeptide trap of item 32, wherein said NRP1 polypeptide corresponds to soluble NRP1 isoform 2.

34. The NRP1 polypeptide trap of item 33, wherein said soluble NPR1 isoform 2 comprises or consists essentially of a polypeptide having an amino acid sequence as set forth in FIG. 22 without a signal peptide.

35. The NRP1 polypeptide trap of item 34, wherein said NRP1 polypeptide corresponds to the extracellular domain of an NRP1 isoform 1 polypeptide.

36. The NRP1 polypeptide trap of item 35, wherein said NRP1 isoform 1 polypeptide is as set forth in FIG. 26 and wherein said extracellular domain corresponds to amino acids 22 to 859.

37. The NRP1 polypeptide trap of any one of items 32 to 36, wherein said NRP1 trap comprises an NRP1 polypeptide comprising (i) amino acids 22 to 609 of a NRP1 polypeptide as set forth in SEQ ID NO: 65; (ii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 66; (iii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 69 (iv) or a functional fragment or functional variant of (i), (ii) or (iii).

38. The NRP1 polypeptide trap of item any one of items 32 to 37, wherein NRP1 trap binds to SEMA3A but does not substantially bind to VEGF165 or has a reduced binding affinity for VEGF165 as compared to SEMA3A binding affinity.

39. The NRP1 polypeptide trap of item 38, wherein said NRP1 trap (i) lacks completely or partially domain b1 and/or b2 of NRP1; or (ii) comprises at least one amino acid point mutation which inhibits VEGF binding to NRP1.

40. The NRP1 polypeptide trap of item 39, wherein said point mutation comprises (a) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to tyrosine 297 of an NRP1 amino acid sequence set forth in FIG. 22 or FIG. 26; (b) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to aspartic acid 320 of an NRP1 amino acid sequence set forth in FIG. 22 or FIG. 26; and/or (c) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to glutamic acid 319 of an NRP1 amino acid sequence set forth in FIG. 22 or FIG. 26.

41. The NRP1 polypeptide trap of item 40, wherein said mutation point is a Y297A substitution; a D320K substitution and/or a E319K substitution.

42. The NRP1 polypeptide trap of any one of items 32 to 38, wherein said trap: (a) comprises domains a1, a2, b1, b2, and c and of said NRP1 polypeptide; (b) comprises domains a1, a2, b1 and b2 of said NRP1 polypeptide; (c) comprises domains a1, a2, and b1 of said NRP1 polypeptide; (d) comprises domains a1 and a2 said NRP1 polypeptide; (e) comprises domain b1 of said NRP1 polypeptide, wherein said domain b1 comprises at least one point mutation at an amino acid residue corresponding to (i) tyrosine 297; (ii) aspartic acid 320 and/or (iii) glutamic acid 319, of a NRP1 polypeptide comprising an amino acid sequence as set forth in FIG. 26, wherein said at least one mutation reduces or abrogates binding to VEGF165; (f) lacks completely or partially domain c of said NRP1 polypeptide; (g) lacks completely or partially domain b1 of said NRP1 polypeptide; (h) lacks completely or partially domain b2 of said NRP1 polypeptide; (i) lacks domains b1 and b2 of said NRP1 polypeptide; or (j) lacks domains b1, b2 and c of said NRP1 polypeptide.

43. The NRP1 polypeptide trap of item 42, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 27 to 141 of an NRP1 polypeptide as set forth in FIG. 26; (ii) said domain a2 comprises an amino acid sequence corresponding to amino acids 147 to 265 of an NRP1 polypeptide as set forth in FIG. 26; (iii) said domain b1 comprises an amino acids sequence corresponding to amino acids 275 to 424 of an NRP1 polypeptide as set forth in FIG. 26; (iv) said domain b2 comprises an amino acids sequence corresponding to amino acids 431 to 583 of an NRP1 polypeptide as set forth in FIG. 26; and/or (v) said domain c domain comprises an amino acids sequence corresponding to amino acids 645 to 811 of an NRP1 polypeptide as set forth in FIG. 26 [correspond à la definition alternative des différents domaines transmis par N. Beaulieu—SEQ ID NOs: 71-75].

44. The NRP1 polypeptide trap of item 42, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 22 to 148 of an NRP1 polypeptide as set forth in FIG. 26; (ii) said domain a2 comprises an amino acid sequence corresponding to amino acids 149 to 275 of an NRP1 polypeptide as set forth in FIG. 26; (iii) said domain b1 comprises an amino acids sequence corresponding to amino acids 276 to 428 of an NRP1 polypeptide as set forth in FIG. 26; (iv) said domain b2 comprises an amino acids sequence corresponding to amino acids 429 to 589 of an NRP1 polypeptide as set forth in FIG. 26; and/or (v) said domain c domain comprises an amino acids sequence corresponding to amino acids 590 to 859 of an NRP1 polypeptide as set forth in FIG. 26.

45. The NRP1 polypeptide trap of item 32, wherein said trap consists essentially of a trap as set forth in Table 1 or a functional variant thereof.

46. The NRP1 polypeptide trap of any one of items 32 to 44, wherein said trap further comprises a protein purification domain.

47. The NRP1 polypeptide trap of item 46, wherein said purification domain is a polyhistidine tag.

48. The NRP1 polypeptide trap of any one of items 32 to 47, wherein said NRP1 trap further comprises a FC domain.

49. The NRP1 polypeptide trap of any one of items 46 to 48, wherein said NRP1 trap comprises a protease or peptidase cleavage site enabling said protein purification domain or FC domain to be removed from said NRP1 trap.

50. The NRP1 polypeptide trap of item 49, wherein said protease or peptidase cleavage site is a TEV protease cleavage site.

51. The NRP1 polypeptide trap of item 50, wherein said TEV protease cleavage site comprises the amino acid sequence GSKENLYFQG.

52. A nucleic acid encoding the NRP1 polypeptide trap of any one of items 32-51.

53. An expression vector comprising the nucleic acid of item 52.

54. A host cell comprising the vector of item 53.

55. A composition comprising the NRP1 polypeptide trap of any one of items 32 to 51, the nucleic acid of item 52, the vector of item 53 or the host cell of item 54 and a suitable carrier.

56. The composition of item 55 for (ii) for preventing or treating inflammation, or (ii) preventing or reducing hyperactivation of innate immune response.

57. A compound for preventing or treating inflammation, wherein said compound: (a) reduces NRP1 expression or activity; and/or (b) reduces NRP1 ligand expression or activity.

58. A compound for preventing or reducing hyperactivation of innate immune response, wherein said compound: (a) reduces NRP1 expression or activity; and/or (b) reduces NRP1 ligand expression or activity.

59. The compound of item 57 or 58, wherein said compound is: (i) A anti SEMA3A antibody; (ii) An anti VEGF165 antibody; (iii) A anti NRP1 antibody (iv) A NRP1 trap; (v) A SEMA3A antisense, shRNA or siRNA; (vi) A NRP1 antisense, shRNA or siRNA; or (vii) A VEGF antisense, shRNA or siRNA.

60. The compound item 59, wherein said compound is an NRP1 polypeptide trap.

61. A composition for treating or preventing inflammation comprising a compound as defined in any one of items 57-60 and a suitable carrier.

62. A composition for preventing or reducing hyperactivation of innate immune response comprising a compound as defined in of any one of items 57-60 and a suitable carrier.

63. Use of the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound of any one of items 57-60 or the composition of any one of items 55, 61 and 62 in the manufacture of a medicament for preventing or treating inflammation.

64. Use of the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound of any one of items 57-60 or the composition of any one of items 55, 61 and 62 in the manufacture of a medicament for preventing or treating inflammation.

65. Use of the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound as defined in any one of items 57-60 or the composition as defined in any one of items 55, 61 and 62 for preventing or treating hyperactivation of innate immune response.

66. Use of a the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound as defined in any one of items 57-60 or the composition as defined in any one of items 55, 61 and 62 for preventing or reducing hyperactivation of innate immune response.

67. The method of any one of items 1-31, wherein said subject suffers or is likely to suffer from septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis, diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease or stroke.

68. The method of any one of items 1-31, the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54, a compound as defined in any one of items 57-60 or a composition as defined in any one of items 55, 61 and 62 wherein said method, NRP1 polypeptide trap, nucleic acid, vector, host cell, compound, composition or use is for treating or preventing septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis, diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease or stroke.

69. The method of any one of items 1-31, the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54, a compound as defined in any one of items 57-60 or a composition as defined in any one of items 55, 61 and 62 wherein said method, NRP1 polypeptide trap, nucleic acid, vector, host cell, compound, composition or use is for treating or preventing septic shock or stroke.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

In the appended drawings:

FIG. 1 shows that NRP1 identifies a population of microglia that is mobilized secondary to vascular injury. (A). Schematic depiction of the mouse model of oxygen-induced retinopathy (OIR). The first phase (postnatal day 7-12 (P7-P12)), under 75% oxygen, induces vasoobliteration. The second phase (under room air) from postnatal day 12 to 17 (P7-P17) allows to attain maximal pre-retinal neovascularization. (B, E and H) show representative FACS plots of CD11b+/F4−80+/Gr-1⁻ cells (microglia) in retinas collected at P10 (B), P14 (E) and P17 (H) from WT OIR and Normoxic control mice. (C, F and I) shows the fold change in the number of retinal microglia in Normoxia (N) and OIR at P10 (C), P14 (F) and P17 (I). The number of retinal microglia was significantly increased in OIR at all points analyzed (C, F and I); n=7-8 (Normoxia, (N)), n=7-8 (OIR) (total of 28-32 retinas per condition; each “n” comprises 4 retinas). (D, G and J) show the fold change in the number of NRP1 positive MPs at P10 (D), P14 (G) and P17 (J). A proportional increase in the number of NRP1-positive microglia was observed in OIR retinas (D, G and J); n=3-5 (Normoxia, (N)), n=3-5 (OIR) (total of 12-20 retinas per condition; each “n” is comprised of 4 retinas). (K). To investigate the role of MP-resident NRP1, LysM-Cre/Nrp1^(fl/fl) mice which have significantly compromised NRP1 expression in retinal microglia were generated (n=3 (WT), n=4 (LysM-Cre/Nrp1^(flfl), total of 12-16 retinas per condition). Left panel shows the % of NPR-1 positive MPs in WT (LysM-Cre/NRP1^(+/+)) and mice deficient in NRP1 in their myeloid cells (LysM-Cre/Nrp1^(fl/fl)) as determined by FACS (right panel). (L, N and P), FACS analysis at P10 (L), P14 (N) and P17 (P) to quantify the number of MPs in LysM-Cre/Nrp1^(fl/fl) mice retinas in Normoxia and OIR. (M, O and Q) shows the fold change in the number of MPs in LysM-Cre/Nrp1^(fl/fl) mice retinas in normoxia and OIR. FACS analysis at P10 and P14 during the proliferative phase of OIR (L, N) reveals that MP-resident NRP1 is essential for MP infiltration into the ischemic retina as LysM-Cre/Nrp1^(fl/fl) mice did not show an increase in numbers of CD11b+/F4−80+/Gr-1⁻ cells in OIR at these time points (M, O). At P17, MPs infiltrate independent of NRP1 (P, Q). n=7-8 (N), n=7-9 (OIR) (total of 28-36 retinas per condition; each “n” comprises 4 retinas). (R) Summary graph of MP accumulation in the retina over the course of OIR in WT and LysM-Cre/Nrp1^(fl/fl) mice. (S, T) Representative FACS plots depicting that Gr1⁻/CD11b⁻+/F4/80+ cells express high levels of CX3CR1 and intermediate/low levels of CD45. CX3CR1high and CD45low cells express NRP1 in WT retinas (S) and do not express NRP1 in retinas from LysM-Cre/Nrp1^(fl/fl) mice (T). Data is expressed as fold change relative to control±SEM. *P<0.05, **P<0.001,***P>0.0001;

FIG. 2 shows that NRP1⁺ myeloid cells localize to sites of pathological neovascularization in the retina. Confocal images of Isolectin B4 (vessel and microglia stain) and NRP1-stained retinal flatmounts at P14 with budding neovascular tufts in WT (A) and LysM-Cre/Nrp1^(fl/fl) mice (G) and at P17 with mature tufts in WT (D) and LysM-Cre/Nrp1^(fl/fl) mice (J). High magnification images reveal co-localization of NRP1-positive microglia (IBA1) with both nascent (B) and mature tufts (E) as confirmed by 3D reconstruction (C, D) in WT mice. (C, F, I, L) show 3D reconstruction of tissue. White arrows in (A, right panel) point to sprouting tufts. White arrows in (B, E) point to NRP1⁺ MPs associated with tufts. LysM-Cre/Nrp1^(fl/fl) mice had less MPs and less tufting (G-K). For all IHCs, representative images of three independent experiments are shown. Scale bars (A, D, G, J): 100 μm, (B, E, H, K): 50 μm;

FIG. 3 shows that the NRP1 ligand, SEMA3A, is induced in patients suffering from proliferative diabetic retinopathy. Angiographies, funduscopies, spectral-domain optical coherence tomography (SD-OCT) and three-dimensional (3D) retinal maps obtained from patients selected for the study. Control patients had non-vascular pathologies and were compared to patients with proliferative diabetic retinopathy (PDR). Control ERM patients shows signs of non-diabetes-related retinal damage such as (A, B) tractional tension on vasculature (arrow) secondary to (C) fibrotic tissue (white arrow), posterior vitreous detachment (arrowhead) and macular bulging (angiography and 3D map). Retinas from PDR patients have (E) neovascularization (inset) with (D) highly permeable microvessels as evidenced by leakage of fluorescent dye (inset), (F) microaneurysms (inset arrows) and (G) fibrous scar tissue (arrow), indicative of advanced retinopathy. (H) PDR patients show some evidence of macular edema, including cystoid formation (white arrowhead) due to focal coalescence of extravasated fluid. (I) Vitreous humour analyzed by ELISA shows increased levels of SEMA3A protein by 5-fold in patients with PDR; n=17 for controls and 17 with PDR. (J) Western blot analysis of equal volumes of vitreous corroborates the increase in SEMA3A (˜125 KDa and 95 KDa) in patients with PDR with respect to controls;

FIG. 4 shows that ligands of NRP1 are induced in the retinal ganglion cell layer during OIR. (A, B) Retinas from WT and myeloid deficient NRP1 k.o. mice (LysM-Cre/Nrp1^(fl/fl) mice) under normoxic conditions or in OIR were collected between P10 and P17 and analyzed by RT-qPCR (oligonucleotide used were as disclosed in Example 11, Table 2). SEMA3A mRNA (A) expression was induced throughout OIR in both WT and LysM-Cre/Nrp1^(fl/fl) retinas while VEGF (B) was significantly less induced in k.o. mice (LysM-Cre/Nrp1^(fl/fl)) compared to WT retinas (stars). Data are expressed as a fold change relative to respective normoxic controls for each time point±SEM; n=4-7; *p<0.05, **p<0.01, ***p<0.001. (C) Laser capture micro-dissection (LCM) was performed on P14 mice with care being taken to select avascular retinal zones in OIR. (D, E) RT-qPCR on LCM of retinal layers in control and OIR avascular zones showed an induction in both SEMA3A (D) and VEGF (E) mRNA in the ganglion cell layer (GCL) during OIR retinas compared to normoxic retinas. VEGF was also induced in the inner nuclear layer of OIR retinas (E). Data are expressed as a fold change relative to normoxic GCL±SEM;

FIG. 5 shows that NRP1⁺ MPs do not proliferate in the retina after vascular injury. Representative FACS histograms of CD11b+/F4−80+/Gr-1⁻ cells obtained from retinas (A) and spleens (B) collected at P14 from WT OIR (right panel) and Normoxic (left panel) control mice injected with BrdU at P13. The number of BrdU⁺ cells was considerably higher in spleens but did not change significantly between OIR and Normoxic mice (C). n=4 (Normoxic, N), n=4 (OIR) (total of 16 retinas per condition; each “n” is comprised of 4 retinas). Data are expressed as a percentage of BrdU+ Gr-1⁻/CD11b+/F4−80+ cells±SEM;

FIG. 6 shows that SEMA3A and VEGF are chemo-attractive towards macrophages via NRP1. (A, B) Primary macrophages were isolated from WT or myeloid-deficient NRP1 k.o. mice (LysM-Cre/Nrp1^(fl/fl) mice) and subjected to a transwell migration assay with vehicle, MCP-1 (100 ng/ml), SEMA3A (100 ng/ml) or VEGF (50 ng/ml) added to the lower chamber. Representative images of migrated cells stained with DAPI are shown (A). SEMA3A or VEGF promoted macrophage migration to similar extents as the positive control MCP-1 (B). To ascertain that SEMA3A and VEGF were stimulating macrophage chemotaxis, cells were pre-treated with the selective ROCK inhibitor Y-27632 (100 μg/ml) (B) which abolished chemotaxis. Macrophages from LysM-Cre/Nrp1^(fl/fl) mice were unresponsive to SEMA3A or VEGF but responsive to MCP-1 (C). Data are expressed as a fold change relative to control (non-treated cells); n=6-22; **p<0.01, ***p<0.001. Scale bars: 100 μm (A);

FIG. 7 shows that Nrp1⁺ macrophages promote microvascular growth in ex vivo choroid explants. (A) Quantification and representative images of choroid explants isolated from LysM-Cre/Nrp1^(+/+) and LysM-Cre/Nrp1^(fl/fl) mice (n=6; p=0.018). (B, C) Representative images of choroid explants from LysM-Cre/Nrp1^(+/+) (B) and LysM-Cre/Nrp1^(fl/fl) (C) mice following chlodronate liposome treatment (to deplete macrophages) and subsequent addition of exogenous macrophages (Ma). (D, E) Quantification of choroidal microvascular sprouting from LysM-Cre/Nrp1^(+/+) (D) and LysM-Cre/Nrp1^(fl/fl) (E) depicted in B and C (n=6, n.s.: not significant, * p<0.05, **p<0.01, ***p<0.001);

FIG. 8 shows that deficiency in myeloid-resident NRP1 reduces vascular degeneration and pathological neovascularization in retinopathy. Wild-type, LysMCre/Nrp1^(+/+) and LysM-Cre/Nrp1^(fl/fl) mice were subjected to OIR and retinas collected at P12 and P17, flatmounted and stained with Isolectin B4. LysM-Cre/Nrp1^(fl/fl) mice had less vasoobliteration at P12 (#3 in A, B) and reduced avascular areas (#3 in C, D) and preretinal neovascularization (#3 in E, F) at P17 compared to both control WT (#1) or control LysMCre/Nrp1^(fl/fl) mice (#2). Results are expressed as percentage of avascular or neovascular area versus the whole retinal area; n=5-19. Scale bars: B&D: 1 mm and F: 500 μm. **p<0.01, ***p<0.001;

FIG. 9 shows that therapeutic intravitreal administration of soluble NRP1 reduces MP infiltration and pathological neovascularization in retinopathy. WT mice were subjected to OIR and injected intravitreally at P12 with soluble recombinant mouse NRP1 (rmNRP1 comprising domains a1, a2, b1, b2 and c, see also FIGS. 19C and 20R) as a trap to sequester OIR-induced ligands of NRP1. At P14, FACS analysis revealed a decrease of over 30% in the number of retinal MPs in rmNRP1 injected retinas (A). Data are expressed as a fold change relative to control (vehicle-injected retinas)±SEM; n=3-4 (total of 12-16 retinas per condition; each “n” comprises 4 retinas). Treatment with rmNRP1 efficiently decreased pathological neovascularization at P17 when compared to vehicle-injected eyes (B, C). Results are expressed as percentage of neovascular area versus the whole retinal area; n=11. Scale bars: 500 μm. *p<0.05, **p<0.01;

FIG. 10 is a schematic depiction of the instant findings illustrating that during ischemic retinopathies such as that of diabetes, avascular zones of the retina, ischemic neurons and neural tissue produces ligands of NRP1 (SEMA3A and VEGF), which in turn act as potent chemo-attractive agents for pro-angiogenic microglia. The NRP1⁺ microglia then partake in the pathogenesis of proliferative retinopathy;

FIG. 11 shows that SEMA3A is upregulated in several organs during septic shock. mRNA levels of SEMA3A (left panels) and VEGF (right panels) were assessed by qRT-PCR following LPS-induced (15 mg/kg) sepsis in mice. SEMA3A and VEGF mRNA levels were normalized with β-actin expression and fold changes in mRNA levels were determined at 0, 6, 12 and 24 hours following LPS administration. A. Fold change in SEMA3A (left panel) and VEGF (right panel) in mice brain. B. Fold change in SEMA3A (left panel) and VEGF (right panel) in mice kidneys. C. Fold change in SEMA3A (left panel) and VEGF (right panel) in mice lungs. D. Fold change in SEMA3A (left panel) and VEGF (right panel) in mice liver;

FIG. 12 shows cytokines expression following LPS-induced sepsis. mRNA levels of TNF-α and IL-13 were assessed by qRT-PCR following LPS-induced sepsis (15 mg/kg) in mice. mRNA levels were normalized with β-actin expression an fold changes in mRNA levels were determined at 0, 6, 12 and 24 hours following LPS administration. A. Fold change in TNF-α (left panel) and IL-1β (right panel) in mice brain. B. Fold change in TNF-α (left panel) and IL-1β (right panel) in mice kidneys. C. Fold change in TNF-α (left panel) and IL-1β (right panel) in mice lungs. D. Fold change in TNF-α (left panel) and IL-1β (right panel) in mice liver;

FIG. 13 shows that SEMA3A induces secretion of pro-inflammatory cytokines in myeloid cells via NRP1. Wild-type and NRP1 knock out (LyzM/NRP1^(fl/fl)) myeloid cells were treated with SEMA3A (100 ng/nml) or vehicle and IL-6 (A), TNF-α (B) and IL-1β (C) protein secretion was analyzed by Cytometric Bead Array (CBA);

FIG. 14 shows that myeloid deficiency in NRP1 reduces production of inflammatory cytokines during sepsis in vivo. NRP1 knock out mice (LyzM/NRP1^(fl/fl)) and control wild type mice were administered vehicle or LPS (15 mg/kg) to induce sepsis. Brains and livers were collected 6 hours post LPS injection and mRNA extracted. TNF-α (A, C) and IL-1β (B, D) expression was analyzed by real-time RT-PCR and levels normalized with β-actin expression level;

FIG. 15 shows that in vivo inhibition of NRP1 activity prevents sepsis-induced barrier function breakdown. Mice were administered with i) vehicle, ii) LPS (15 mg/kg); or iii) LPS (15 mg/kg) and an NRP1 trap (Trap-1, FIGS. 19C and 20R but without an FC domain, NP_032763, 4 ug/0.2 mg/kg, i.v.). Vascular permeability in brain (A), kidney (B) and liver (C) was then assessed using an Evan blue permeation assay (EBP);

FIG. 16 shows that in vivo inhibition of NRP1 activity protects against sepsis. (A) Survival rate of control mice administered with i) a high dose of LPS (i.p., 25/mg/kg); or ii) an NRP1 trap (i.v., 0.2 mg/kg of Trap-1, FIGS. 19C and 20R but without an FC domain, NP_032763) followed by a high dose of LPS (i.p., 25/mg/kg). (B) Comparison of survival rate between myeloid-resident NRP1 knock out mice (LyzM/NRP1^(fl/fl)) and control mice administered with a high dose of LPS (i.p., 25/mg/kg);

FIG. 17 shows that administration of NRP1 derived trap or myeloid deficiency in NRP1 lowers inflammatory cytokine production in septic shock. Wild-type mice were administered i) vehicle (n=3), ii) LPS (15 mg/kg, n=3) or iii) LPS and an NRP1 trap (Trap-1, FIGS. 19C and 20R but without an FC domain, NP_032763). Mice with NRP1 deficient myeloid cells (LyzM-Cre/Nrp^(fl/fl)) were administered LPS (15 mg/kg, n=3). Brains were collected 6 hours post LPS injection and production of TNF-α (A) and IL-6 (B) was measured;

FIG. 18 shows that administration of NRP1 derived trap protects against ischemic stroke. Mice were subjected to transient middle cerebral artery occlusion (MCAO) and administered vehicle or NRP1 trap and the size of the infarct (stroke) measured on coronal cerebral sections stained with cresyl violet. The unstained area corresponds to the damaged area. (A), Coronal cerebral sections of MCAO mice treated with vehicle. (B) Coronal cerebral sections of MCAO mice treated with NRP1 Trap-1 (see FIGS. 19C and 20R but without an FC domain, NP_032763). (C) Schematic representation of average infarct size in mice treated with vehicle or NRP1 trap following MCAO. (D) Neurological impairment (neuroscore) of mice treated with vehicle or NRP1 trap 1 h after MCAO. (E) Neurological impairment (neuroscore) of mice treated with vehicle or NRP1 trap 24 h after MCAO;

FIG. 19 shows a schematic representation of the NRP1 protein and embodiments of NRP1-traps of the present invention. (A). WT NRP1 representation showing SEMA3A binding domain (mainly a1a2 with a small contribution of b1 and VEGF binding domain (b1b2). The c-domain is the MEM domain that is thought to contribute to NRP dimerization to other co-receptors. (B-F) Schematic representations of human-derived NRP1 (C) and mouse-derived NRP1 traps;

FIG. 20 shows the nucleic acid and protein sequences of the NRP1 traps depicted in FIGS. 19B and C. (A) Trap 1/TrappeA—full NRP1-FC amino acid (SEQ ID NO: 114) and nucleotide (SEQ ID NO: 2) sequences; (B) Trap 2—NRP1-FC-Δ c-amino acid sequence (SEQ ID NO: 115); (C) Trap 2—NRP1-FC-Δ c-nucleotide sequence (SEQ ID NO: 4); (D) Trap 3—NRP1-FC-Δ b2c-amino acid sequence (SEQ ID NO: 116); (E). Trap 3—NRP1-FC-Δ b2c-nucleotide sequence (SEQ ID NO: 6); (F) Trap 4—NRP1-FC-Δb1 b2c-amino acid sequence (SEQ ID NO: 117); (G) Trap 4—NRP1-FC-Δb1 b2c-nucleotide sequence (SEQ ID NO: 8); (H) Trap 5/Trap I—NRP1-FC Δ c-short-amino acid (SEQ ID NO: 118) and nucleotide (SEQ ID NO: 10) sequences; (I) Trap 6/Trap D—NRP1-FC Δ b2c-short-amino acid (SEQ ID NO: 119) and nucleotide (SEQ ID NO: 12) sequences; (J) Trap 7/Trap C—NRP1-FC Δ b1b2c-short-amino acid (SEQ ID NO: 120) and nucleotide (SEQ ID NO: 14) sequences; (K) Trap 8/TrapJ—full NRP1-FC-VEGF low-amino acid (SEQ ID NO: 121) and nucleotide (SEQ ID NO: 16) sequences; (L) Trap 9—NRP1-FC-Δ c-VEGF low-amino acid sequence (SEQ ID NO: 122); (M) Trap 9—NRP1-FC-Δ c-VEGF low-nucleotide sequence (SEQ ID NO: 18); (N) Trap 10—NRP1-FC-Δ b2c-VEGF low-amino acid sequence (SEQ ID NO:123); (O) Trap 10—NRP1-FC-Δ b2c-VEGF low-nucleotide sequence (SEQ ID NO: 20); (P) Trap 11/TrapL—NRP1-FC-Δ c-VEGF low—Short-amino acid (SEQ ID NO: 124) and nucleotide (SEQ ID NO:22) sequences; (Q) Trap 12/TrapK—NRP1-FC-Δb2 c-VEGF low—Short-amino acid (SEQ ID NO:125) and nucleotide (SEQ ID NO:24) sequences. (R) Mouse Trap 1—full Nrp1-mFC amino acid (SEQ ID NO: 126) and nucleotide (SEQ ID NO: 26) sequences. (S) Mouse Trap 2—Nrp1-mFC Δ c-short amino acid (SEQ ID NO: 127) and nucleotide (SEQ ID NO: 28) sequences. (T) Mouse Trap 3—Nrp1-FC Δ b2c-short amino acid (SEQ ID NO: 128) and nucleotide (SEQ ID NO: 30) sequences;

FIG. 21 shows human SEMA3A precursor protein sequence (SEQ ID NO: 31). This sequence is further processed into mature form. Residues 1-20 correspond to the signal peptide;

FIG. 22 shows human soluble Neuropilin-1 (NRP1) receptor protein sequence (e.g., GenBank Acc. No. AAH07737.1-SEQ ID NO: 32). Domains a1, a2, B1, b2 and c are shown. Domain a1 consist of amino acids 23-148; domain a2 consist of amino acids 149-270; domain b1 consist of amino acids 271-428; domain b2 consists of amino acids 429-590 and domain c consists of amino acids 591-609;

FIG. 23 shows that SEMA3A traps accelerate vascular regeneration and reduce pathological angiogenesis in ischemic mice retinas in an oxygen-induced retinopathy model. (A) Schematic depiction of the mouse model of oxygen-induced retinopathy (OIR) showing the four principal stages of retinopathy i.e., normoxia, vessel loss/vaso-obliteration, proliferation/neovascularization and neovascular (NV) regression. (B) Mean percentage (%) of avascular area (relative to vehicle) at P17 following intravitreal injection of histidine tagged Trap G or Trap M (Trap G-HIS (SEQ ID NO: 38) and TrapM-HIS (SEQ ID NO: 42)). Photographs of representative retinas showing avascular area are shown for each group. (C) Mean percentage of neovascular area (relative to vehicle) at P17 following intravitreal injection of histidine tagged Trap G and Trap M. Photographs of representative retinas showing neovascular area are shown for each group. *p<0.05, **p<0.01, ***p<0.001. n=8-13 animals/group;

FIG. 24 shows that SEMA3A trap prevents vascular leakage and edema in diabetic retinas. (A). Blood glucose levels of mice prior streptozotocin (STZ) treatment (week 0) and 3 weeks following STZ treatment (diabetic state). (B). Retinal Evans Blue permeation assay (measured at week 8) on mice retinas injected intravitreally with 0.5 ug/ml of Trap G, Trap M or 80 μg (1 ul) of anti-VEGF₁₆₄ antibody (AF-493-NA, R&D) at 6 and 7 weeks following STZ administration. (C) Retinal Evans Blue permeation assay (measured at week 14) on mice retinas injected intravitreally with 0.5 ug/ml of Trap G or Trap M or anti-VEGF₁₆₄ antibody (AF-493-NA, Novus Biologicals) at 12 and 13 weeks post STZ treatment. *p<0.05, n=4, from 12 animals;

FIG. 25 shows that NRP1 derived trap (anti SEMA3A and VEGF) reduces choroidal neovascularization in a model of age-related macular degeneration (AMD) (A). Schematic representation of the method used for inducing choroidal neovascularization in mice eyes. (B) Choroidal Neovascularization at day 14 post laser burn (mean perfused FITC/Lectin area). Mice eyes were injected intravitreally with Trap G right after laser burn:

FIG. 26 shows an alignment between rat (Access. Nos. EDL96784, NP_659566), human (SEQ ID NO: 68, Accession No. NM003873) and mouse (SEQ ID NO: 67, Accession No. NP_032763) together with an NRP1 consensus sequence (SEQ ID NO: 69). The NRP1 signal domain (amino acids 1-21), SEMA3a binding domains a1 (amino acids 22-148, SEQ ID NO:78), a2 (amino acids 149-175, SEQ ID NO:79), VEGF binding domains b1 (amino acids 276-428, SEQ ID NO:80) and b2 (amino acids 429-589, SEQ ID NO:81), domain c (amino acids 590-859, SEQ ID NO:82), transmembrane domain (amino acids 860-883, SEQ ID NO:77) and cytoplasmic domain (amino acids 884-923) are identified; and

FIG. 27 shows protein sequence alignments between exemplary traps of the present invention shown in FIG. 19 but without any histidine or FC tags. (A). protein sequence alignment between exemplary traps but lacking the 6XHis tag purification domains (G (SEQ ID NO:100), R (SEQ ID NO:101), Z (SEQ ID NO:102), AB (SEQ ID NO:103), AC (SEQ ID NO:104), 0 (SEQ ID NO:105), Q (SEQ ID NO:106), M (SEQ ID NO:107), P (SEQ ID NO:108), N (SEQ ID NO:109), W (SEQ ID NO: 110), X (SEQ ID NO: 111) and Y (SEQ ID NO: 112)) of the present invention comprising a 6X His tag purification domain. (B) protein sequence alignment between exemplary traps of the present invention but lacking the FC domain ((A (SEQ ID NO:100), I (SEQ ID NO:105), D (SEQ ID NO:107), C (SEQ ID NO:109), J (SEQ ID NO:101), L (SEQ ID NO:106), K (SEQ ID NO:108), S (SEQ ID NO:113), U (SEQ ID NO:111), V (SEQ ID NO:112)).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present inventors have identified a subset of mononuclear phagocytes (MPs) that responds to local chemotactic cues such as SEMA3A that are conserved between central neurons, vessels and immune cells. NRP1 expressing MP's were shown to enter the site of injury and to contribute to (i) tissue damage and/or (ii) pathological activation of the innate immune response in models of inflammatory conditions including various forms of inflammatory, proliferative retinopathies (e.g., proliferative diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration), septic shock and cerebral ischemia/stroke.

The inventors demonstrated that stressed retinal neurons and neural tissue have the inherent ability to modulate the local innate immune response via unconventional chemotactic agents. NRP1 on microglia was found to be a potent chemoattractive receptor for SEMA3A, and VEGF and inhibition of NRP1 signaling in innate immune cells (e.g., using NRP1-derived traps or NRP1 or SEMA3A antibodies) resulted in protection against MP's induced inflammation and tissue damage.

Patients suffering from late stage proliferative diabetic retinopathy (PDR) were shown to produce elevated levels of SEMA3A which counterintuitively acts as a potent attractant for Neuropilin-1 (NRP1)-positive MPs. These pro-angiogenic MPs are selectively recruited to sites of pathological neovascularization in response to locally produced SEMA3A as well as VEGF and TGF-β. Furthermore, SEMA3A was shown to be up-regulated in several organs during septic shock and to induce secretion of inflammatory cytokines by MP's. Inhibition of NRP1 also reduced the production of proinflammatory cytokines in sepsis.

Finally NRP1-positive MPs were shown to play a critical role in inflammatory disease progression. Inhibition/abrogation of NRP1 myeloid-dependent activity was shown to protect against neovascular retinal disease (vascular degeneration and pathological neovascularization), septic shock and neural damages secondary to cerebral ischemia/stroke.

Together, these findings underscore the role of NRP1-positive MPs and their ligands in inflammation (and in particular in neuroinflammation) and demonstrate the therapeutic benefit of inhibiting NRP1 cell signaling to limit hyperactivation of innate immune response (e.g., tissue damage at the site of injury through recruitment of microglia/macrophages and/or induction of production and/or secretion of proinflammatory cytokines, and/or vascular leakage/edema). The present findings finds applications in the prevention and treatment of diseases and conditions characterized by sustained (e.g., chronic, persistent) or excessive/pathological inflammation involving MP recruitment and proinflammatory cytokines production and secretion such as septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis and neuroinflammatory conditions such as diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease and stroke.

Inhibition of NRP1-Mediated Cellular Activity

The present inventors have found that by inhibiting NRP1-dependent cell signaling (and in particular SEMA3A-mediated cell signaling), it is possible to protect against (prevent or treat) inflammatory diseases and conditions such as those involving hyperactivation of the innate immune response. In particular, inhibition of NRP1-mediated cell-signaling reduces the unwanted (pathological) recruitment of mononuclear phagocytes (MPs, e.g., microglia, macrophages) and the production/secretion of proinflammatory cytokines which contribute to tissue damage (e.g., increased vascular degeneration, pathologic neovascularization, cell death or cell damages), inflammation and edema.

Thus, in an aspect, the present invention relates to a method of treating or preventing inflammation comprising inhibiting NRP1-dependent cell-signaling. In a particular aspect, the inflammation is neuroinflammation.

As used herein, the term “inflammation” means a disease or condition which involves the activation of the innate immune response comprising i) the recruitment of mononuclear phagocytes (e.g., microglia or macrophages) expressing the NRP1 receptor at the site of inflammation or injury; and/or ii) the NRP1 dependent production/secretion of pro-inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6). The classical signs of acute inflammation are pain, heat, redness, swelling, and loss of function. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged (sustained) inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Non-limiting examples of inflammatory conditions which may be treated or prevented in accordance with methods of the present invention include septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis and neuroinflammatory conditions such as diabetic retinopathy (including proliferative diabetic retinopathy (PDR)), age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease and stroke.

In a particular embodiment the inflammatory disease or condition is not a retinopathy. In another embodiment, the inflammatory disease or condition is not diabetic retinopathy. In another embodiment, the inflammatory disease or condition is not macular edema. In another embodiment, the inflammatory disease or condition is not diabetic macular edema.

In a related aspect, the present invention concerns a method of inhibiting hyperactivation (or pathological activation) of the innate immune response comprising inhibiting NRP1-dependent cell-signaling. Such an hyperactivation of innate immune response, is typically associated with acute or chronic activation of any given cell population of the immune system (innate and adaptive, e.g., mononuclear cell recruitment in the organ/tissue) beyond levels required to maintain tissue homeostatsis. This is often accompanied by heightened production of cytokines (e.g., TNF-alpha, IL-6), increased vascular permeability, and may result in compromised tissue function.

In another aspect, the present invention concerns a method of treating or preventing vascular degeneration comprising inhibiting NRP1-dependent cell-signaling.

In a further aspect, the present invention concerns a method of treating or preventing pathological neovascularization comprising inhibiting NRP1-dependent cell-signaling.

In another aspect, the present invention concerns a method of treating or preventing septic shock comprising inhibiting NRP1-dependent cell-signaling.

In a yet another aspect, the present invention concerns a method of treating or preventing neural damages secondary to cerebral ischemia/stroke comprising inhibiting NRP1-dependent cell-signaling.

Because NRP1-mediated cell signaling (e.g., MPs recruitment and production/secretion of pro-inflammatory cytokines) depends on the binding of NRP1 to its ligands (e.g., SEMA3A, VEGF and/or TGF-β), inhibition of NRP1-mediated cellular signaling can be achieved in at least two ways: i) by targeting the expression or activity of NRP1 directly (through the use of NRP1 antibodies, NRP1 derived traps or the like); or ii) by targeting the expression or activity of one or more of its ligands (e.g., SEMA3A, VEGF and/or TGF-β).

In embodiments, the above methods comprise preferentially or specifically inhibiting SEMA3A-mediated cell signalling. “Preferentially inhibiting” means that the level of inhibition of SEMA3A-mediated cell signalling is greater than that of other NRP1 ligands (e.g., VEGF165 and TGF-beta). In certain aspects, methods of the present invention substantially do not reduce or inhibit VEGF (e.g., VEGF165) and/or TGF-beta-mediated cell signalling that occur through the interaction with NRP1. In embodiments, compounds of the present invention (e.g., NRP1 traps) “preferentially bind” to one ligand over the others (e.g., preferentially bind SEMA3A over VEGF). Such preferential interaction may be determined by measuring the dissociation constant (Kd) for each ligand. In embodiments, interaction for one ligand (e.g., SEMA3A) over the others (e.g., VEGF) is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 28, 20, 22, 25, 30, 35, 40, 45, 50, 60, 75, 80, 100, 200, 300, 400, 500, 1000 times greater or more. In embodiments the kD (e.g., in nM) for one ligand is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 28, 20, 22, 25, 30, 35, 40, 45, 50, 60, 75, 80, 100, 200, 300, 400, 500, 1000 times smaller than the kD for one or more of the other ligands (e.g., VEGF).

In an embodiment, methods of the present invention comprise administration to a subject likely to suffer from inflammation (e.g., likely to suffer from an inflammatory disease or condition). In other embodiment, methods of the present invention comprise administration to a subject diagnosed from inflammation (e.g., likely to suffer from an inflammatory disease or condition). In an embodiment, the subject is a mammal, preferably a human.

In particular embodiments, compounds used in accordance with the methods

NRP1 Traps

Inhibition of NRP1-mediated cellular signaling can be achieved using NRP1 traps of the present invention. As used herein, the terms, “NRP1 trap”, or “NRP1 polypeptide trap” encompass naturally occurring soluble NRP1 polypeptide (e.g., such as NRP1 secreted isoform b FIG. 22, SEQ ID NO: 65)), and synthetic (e.g., recombinantly produced) NRP1 polypeptide traps including any functional soluble fragment of NRP1 (e.g., NRP1 isoform 1 or 2) or any functional variant of NRP1 which competes with endogenous NRP1 for ligand binding. In an embodiment, the NRP1 traps of the present invention do not exists in nature (i.e., are not naturally occurring) but are “derived” from naturally occurring NRP1 polypeptides (i.e. they are synthetic; e.g., NRP1 traps comprising the extracellular domain of NRP1 isoform 1 or a fragment or variant thereof). NRP1 traps the present invention initially comprise a signal peptide at their N-terminal end (e.g., amino acids 1-21 (SEQ ID NO: 70) of NRP1 shown in FIG. 26 (e.g., SEQ ID NO:69) which is cleaved upon secretion by the cells. Accordingly, NRP1 polypeptide traps of the present invention lack amino acids 1-21 when administered as purified polypeptides or when prepared as pharmaceutical compositions comprising a purified or substantially pure form. Nucleic acids encoding for NRP1 traps of the present invention (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 34, 32, 34, 36, 39, 41, 43, 45, 47, etc. See also Table 1) comprise a polynucleotide sequence in 5′ which encodes for a signal peptide (first 63 nucleotides encoding for the first 21 amino acids at the N-terminal end) which will allow the NRP1 trap to be synthesized and secreted by the cells. In a particular embodiment, the signal peptide corresponds to the first 20 amino acids of the NRP1 polypeptide set forth in SEQ ID NO: 65 (FIG. 22) or SEQ ID NO: 69 (FIG. 26). NRP1 traps of the present invention encompass functional variants of corresponding “wild-type” NRP1 polypeptides or fragment thereof (e.g., polymorphic variations naturally found in the population).

NRP1 traps of the present invention may or may not comprises further polypeptide domains (e.g., purification domains). Exemplary traps lacking purification domains and comprising only NRP1-derived sequences are shown in FIG. 27. Non-limiting examples of NRP1 traps that may be used in accordance with the present invention are given in FIGS. 19B-F, FIG. 20, FIG. 27 and are listed Table 1 below.

TABLE 1 Exemplary NRP1-derived traps which have been prepared in accordance with the present invention. Trap Description SEQ ID Nos. (aa and nts) Trap 1/A Human, “full” extracellular domain NRP1 SEQ ID NOs: 1, 2, (corresponding to amino acids 22 to 856 of NRP1 100 (aa without sequences shown on FIG. 26)-FC FC, includes SP) Trap 2 Human, NRP1-FC-Δc (275 aa linker) SEQ ID NOs: 3, 4 Trap 3 Human, NRP1-FC-Δb2c (434 aa linker) SEQ ID NOs: 5, 6 Trap 4 Human, NRP1-FC-Δb1b2c (593 aa linker) SEQ ID NOs: 7, 8 Trap 5/Trap I Human, NRP1-FC-Δc-short SEQ ID NOs: 8, 10, 101 (aa, without FC, includes SP) Trap 6/TrapD Human, NRP1-FC-Δb2c-short SEQ ID NOs: 11, 12, 107, (aa, without FC, includes SP) Trap 7/TrapC Human, NRP1-FC-Δb1b2c-short SEQ ID NOs: 13, 14 109, (aa, without FC, includes SP) Trap 8/TrapJ Human, “full” extracellular domain NRP1-FC-VEGF SEQ ID NOs: 15, 16, low (Y297A mutation) 101 (aa, without FC, includes SP) Trap 9 Human, NRP1-FC-Δc-VEGF low SEQ ID NOs: 17, 18 (Y297A mutation, 275 aa linker) Trap 10 Human, NRP1-FC-Δb2c-VEGF low SEQ ID NOs: 19, 20 (Y297A mutation, 434 aa linker) Trap 11/Trap L Human, NRP1-FC-Δc-VEGF low-short SEQ ID NOs: 21, 22, (Y297A mutation) 106 (aa, without FC, includes SP) Trap 12Trap K Human, NRP1-FC-Δb2c-VEGF low-short SEQ ID NOs: 23, 24, (Y297A mutation) 108 (aa, without FC, includes SP) mTrap 1 Mouse, “full” extracellular domain NRP1-FC SEQ ID NOs: 25, 26 Amino acids residues 22-856 mTrap 2 Mouse, NRP1-FC-Δc-short SEQ ID NOs: 27, 28 mTrap 3 Mouse, NRP1-FC-Δb2c-short SEQ ID NOs: 29, 30 Trap S Human, NRP1-FC-Δb2-short SEQ ID NOs: 31, 32, 113 (aa without FC, includes SP) Trap U Human, NRP1-FC-Δb2-VEGF low-short SEQ ID NOs: 33, 34, (Y297A mutation) 111 (aa, without FC, includes SP) Trap V Human, NRP1-FC-Δb1b2-short SEQ ID NOs: 35, 36, 112 (aa, without FC, includes SP) Trap G Human, “full” extracellular domain NRP1-His SEQ ID NOs: 38, 39, 100 (aa without his tag, includes SP) Trap O Human, NRP1-His-Δc-short SEQ ID NOs: 40, 41, 105 (aa without FC) Trap M Human, NRP1-His-Δb2c-short SEQ ID NOs: 42, 43, 107 (aa without his tag, includes SP) Trap N Human, NRP1-His-Δb1b2c-short SEQ ID NOs: 44, 45, 109 (aa without his tag, includes SP) Trap R Human, NRP1-His-Δc-VEGF low SEQ ID NOs: 46, 47, 101 (aa without his tag) Trap Q Human, NRP1-His-Δc-VEGF low-short SEQ ID NOs: 48, 49, 106 (aa without his tag, includes SP) Trap P Human, NRP1-His-Δb2c-VEGF low-short SEQ ID NOs: 50, 51, 108 (aa without his tag, includes SP) Trap W Human, NRP1- His-Δb2 -short SEQ ID NOs: 52, 53, 110 (aa without his tag, includes SP) Trap X Human, NRP1- His-Δb2 - VEGF low-short SEQ ID NOs: 54, 55, 111 (aa, without his tag, includes SP) Trap Y Human, NRP1- His-Δb1b2 -short SEQ ID NOs: 56, 57, 112 (aa, without his tag, includes SP) Trap AB Human, “full” extracellular domain NRP1-His- SEQ ID NOs: 58, 59, SEMA3A low (S346A et E348K mutations) 103 (aa, without his tag, includes SP) Trap AC Human, “full” extracellular domain NRP1-His- SEQ ID NOs: 60, 61, VEGF- low (D320K mutation) 104 (aa without his tag, includes SP) Trap Z Human, “full” extracellular domain NRP1-His, SEQ ID NOs: 62, 63, VEGF165-Low (E319K/D320K mutations) 102 (aa, without his tag, includes SP) Trap 1bis Human, Trap 1 without FC SEQ ID NO: 83, 84 SP: Signal peptide

Given that NRP1 distinctly regulates the effects of its ligands on signal transduction and cellular responses, it may be advantageous to specifically inhibit the binding of one specific ligand to NRP1 but not that of the others. For example, as shown herein, at early time points of retinal disease, where SEMA3A levels are elevated, VEGF levels remain low and relatively unchanged compared to non-diabetic controls. Also, in septic shock, SEMA3A was the sole NRP1 ligand which had a long term effect and stayed up-regulated for more than 24 hours following induction of sepsis. Thus, given the differences in expression kinetics for each ligand and the fact that neutralization of one ligand (e.g., VEGF) may be ineffective in certain conditions (or be associated with undesired side effects), specific inhibition of one ligand (e.g., SEMA3A) binding to NRP1, (but not that of the other(s) (e.g., VEGF)) is advantageous. Thus, in certain aspects of the methods of the present invention, inhibition of SEMA3A-mediated cell signaling, is accomplished by providing NRP1 Traps having greater affinity for SEMA3 than VEGF or to which VEGF (e.g., VEGF165) does not bind or does not bind substantially.

Accordingly, in an embodiment, the soluble NRP1 polypeptide or functional fragment or variant thereof (NRP1 trap) of the present invention binds to all natural ligands of NRP1 (e.g., SEMA3A, VEGF and TGF-beta, e.g., a soluble NRP1 trap comprising the extracellular domain (e.g., amino acids 22-856 or 22-959 of SEQ ID NO: 66 or 69), Trap 1, (SEQ ID NO: 1) or Trap G (SEQ ID NO: 38)—See also, FIGS. 19 and 27 and Table 1). In an embodiment, the NRP1-derived trap of the present invention inhibits SEMA3 and VEGF signaling by binding to both SEMA3A and VEGF.

In another embodiment, the NRP1 trap of the present invention is a polypeptide which binds to SEMA3A but not to VEGF. For example the NRP1 trap may comprise the a1 (e.g., SEQ ID NO:71) and/or a2 subdomain(s) (e.g., SEQ ID NO:72) which bind(s) to SEMA3A but not the b1 (e.g., SEQ ID NO:73) and/or b2 (e.g., SEQ ID NO: 74) subdomain(s) required for VEGF binding (e.g., Trap M, (SEQ ID NO: 42), Trap N (SEQ ID NO: 44), Trap 12/Trap K (SEQ ID NO: 23), Trap 4 (SEQ ID NO:7), Trap 7/C (SEQ ID NO: 13), See also, FIGS. 19 and 27 and Table 1). In an embodiment, the NRP1-derived trap comprises domains a1 and a2 corresponding to amino acids 22 to 275 of the NRP1 amino acid sequence set forth in FIG. 26 (e.g., amino acids 22-275 of SEQ ID NO: 66 or SEQ ID NO: 22-275 of SEQ ID NO: 69). The NRP1 trap may also comprise a mutation (e.g., a deletion or substitution) which abrogates or reduces significantly the binding of VEGF to NRP1 but not that of SEMA3A to NRP1 (e.g., Trap 8/trap J (SEQ ID NO:15), Trap 9 (SEQ ID NO: 17), Trap 10 (SEQ ID NO: 19), TRAP 11/L (SEQ ID NO:21), Trap12/K (SEQ ID NO: 23), Trap U (SEQ ID NO: 34), Trap R (SEQ ID NO: 46), Trap Q (SEQ ID NO: 48), Trap P (SEQ ID NO: 50, Trap X (SEQ ID NO:54, Tarp AC (SEQ ID NO: 60), TRAP Z (SEQ ID NO: 62) See also, FIGS. 19 and 27 and Table 1). One non-limiting example of such mutation is a substitution at tyrosine 297 in the b1 domain of NRP1 (e.g., Y297A, FIGS. 19B-D, FIG. 27 and Table 1, e.g., Traps 8, 9, 10, 11, 12, V, R, Q, P and X). Other examples of such mutations comprise a substitution at the glutamic acid at position 319 and at aspartic acid at position 320 in NRP1 (e.g., E319K and D320K such as in Trap AC and Z (SEQ ID NOs: 60, 62)).

In another embodiment, the NRP1 trap is a soluble NRP1 polypeptide or functional fragment or variant thereof which binds to VEGF but not to SEMA3A. For example, the NRP1 trap may comprise the b1 (e.g., SEQ ID NO: 73) and/or b2 (e.g., SEQ ID NO: 74) domain(s) to bind to VEGF but not the a1 (e.g., SEQ ID NO: 71) and/or a2 (e.g., SEQ ID NO: 72) subdomain(s) which bind to SEMA3A. In an embodiment, the NRP1 trap comprises domains b1 b2 corresponding to amino acids 276 to 589 of the NRP1 amino acid sequence set forth in FIG. 26 (e.g., amino acids 276-589 of SEQ ID NO: 66 or 276-289 of SEQ ID NO: 69). In another embodiment, the NRP1 trap may comprise a mutation which reduces or abrogate SEMA3A binding but not that of VEGF. One non-limiting example of such mutation is a substitution at serine 346 and/or glutamic acid 348 of NRP1 (e.g., S346A and E348K mutations, such as in trap AB (SEQ ID NO: 58)—See also FIGS. 19 and 27).

In an embodiment, the soluble NRP1 polypeptide or functional fragment thereof comprises or consists of traps as set forth in FIGS. 19B-F, 20, 27 and Table 1.

In preferred embodiments, the NRP1 traps of the present invention lack the transmembrane domain (e.g., corresponding to amino acids residues 860 to 883 of the NRP1 polypeptide sequences shown in FIG. 26 (such as SEQ ID NO: 66 and 69)) and cytosolic domain (e.g., corresponding to amino acids residues 884-923 of the NRP1 polypeptide isoform 1 sequences shown in FIG. 26 (such as SEQ ID NO: 66 and 69)) found in for example NRP1 isoform 1. In embodiments, the NRP1 traps of the present invention lacks completely or partially domain c of NRP1. NRP1 isoform 1 comprises a larger c domain (see FIG. 26), while that of isoform 2 is shorter (e.g., amino acid sequence VLATEKPTVIDSTIQSGIK (SEQ ID NO: 99) shown in FIG. 22). Particularly, domain c is not essential to SEMA3A and VEGF binding and thus may be excluded from the NRP1 traps used to inhibit NRP1-dependent cell signaling (or SEMA3A-mediated cell signaling). In an embodiment, the NRP1 trap lacks the c domain corresponding to amino acids 590 to 859 of the NRP1 amino acid sequence set forth in FIG. 26 (e.g., amino acids 590 to 859 of SEQ ID NO: 66 or SEQ ID NO: 69). In an embodiment the NRP1 traps of the present invention lack completely or partially the c domain of isoform 2 as set forth in FIG. 22 (e.g., SEQ ID NO: 99). In an embodiment, NRP1 traps of the present invention comprise domain c of NRP1 isoform 2. In another embodiment, the NRP1 derived trap lacks a portion of domain c corresponding to the amino acids set forth in SEQ ID NO: 75.

The soluble NRP1 polypeptide or functional fragment or variant thereof of the present invention may comprise one or more additional polypeptide domain(s) to increase in vivo stability and/or facilitate purification. For example, NRP1 traps of the present invention may include a FC domain (or part thereof such as the human FC domain set forth in SEQ ID NO: 37.) or a purification tag (e.g., a 6X-histidine tag). Such additional polypeptide domain(s) may be linked directly or indirectly (through a linker) to the soluble NRP1 polypeptide or functional fragment thereof.

The soluble NPR1 polypeptide or functional fragment thereof of the present invention may optionally include one or more polypeptide linkers. Such linkers may be used to link one or more additional polypeptide domain(s) to the soluble polypeptide of the present invention (e.g., a polypeptide domain which increases the stability of the polypeptide in vivo and/or a domain which facilitates purification of the polypeptide). Linker sequence may optionally include peptidase or protease cleavage sites which may be used to remove one or more polypeptide fragments or domains (e.g., removal of purification tag prior to in vivo administration of the soluble NRP1 polypeptides or functional fragment thereof). One non-limiting example of a linker or domain which enables cleavage of the polypeptide and removal of, for example, polypeptide domain(s) (e.g., 6X his tag purification domain) includes a polypeptide comprising a TEV protease cleavage site (e.g., GSKENLYFQ′G, SEQ ID NO:76). Many other TEV cleavage sites are known and many other protease/peptidase cleavage sites are known to the skilled person and may be introduced in the polypeptides of the present invention to optionally remove one or more polypeptide domains or fragments.

Polypeptide linkers may also be used to replace totally or partially domains which are normally found in the wild-type NRP1 polypeptide but which are absent in the soluble NRP1 polypeptide or functional fragment thereof of the present invention. For example, in the NRP1 traps of the present invention, synthetic linkers may replace totally or partially domains a1, a2, b1, b2 and c. The length of the linker may correspond to the entire length of the domain removed or to a portion of it. Such linkers may increase protein folding, stability or binding to NRP1 ligands. Non-limiting examples of NRP1 traps comprising linkers are shown in FIGS. 19 and 20 (e.g., Trap 2, Trap 3, Trap 4, Trap 9 and Trap 10 listed in Table 1). One non-limiting example of a useful polypeptide linker is a polyarginine polypeptide. Other linkers are known in the art and may be used in accordance with the present invention.

In an embodiment, the NRP1 trap of the present invention comprises: (i) amino acids 1-856 (preferably, 22 to 856) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (ii) amino acids 1 to 583 (preferably 22 to 583) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (iii) amino acids 1 to 424 (preferably 22-424) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (iv) amino acids 1 to 265 (preferably 22 to 265) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (v) 1 to 430 and 584 to 856 (preferably 22-430 and 584-856) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vi) amino acids 1 to 274 and 584 to 856 (preferably 22-274 and 584 to 856) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vii) amino acids 1 to 430 and 584 (preferably 22 to 430 and 584 to 856) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69). In a particular embodiment, the above noted traps comprise one or more mutation to reduce VEGF or SEMA3A binding as described above.

In a related aspect, the present invention provides nucleic acids encoding the NRP1 traps (e.g., traps listed in Table 1 and shown on FIGS. 19, 20 and 27). Such nucleic acids may be included in an expression vector for expression in cells. Accordingly, the present invention further relates to vectors comprising nucleic acids encoding soluble NRP1 polypeptide or functional fragments thereof and cells comprising such expression vectors. Nucleic acids encoding a soluble NRP1 polypeptide or functional fragment thereof (i.e., NRP-derived trap) of the present invention may include a polynucleotide portion encoding a signal sequence (e.g., encoding amino acids 1-21 of SEQ ID NO: 65, 66 or 69, or SEQ ID NO: 70) for secretion by the cells. Furthermore, nucleic acids of the present invention include nucleic acids with and without a translation termination “stop” codon at their 3′ end. The translation termination stop codon may be provided, for example, by an expression vector into which the nucleic acids of the present invention may be cloned.

As used herein, a “functional fragment” or “functional variant” of NRP1 (e.g., a functional fragment of soluble NRP1 polypeptide or polynucleotide of the present invention such as an NRP1) refers to a molecule which retains substantially the same desired activity as the original molecule but which differs by any modifications, and/or amino acid/nucleotide substitutions, deletions or additions (e.g., fusion with another polypeptide). Modifications can occur anywhere including the polypeptide/polynucleotide backbone (e.g., the amino acid sequence, the amino acid side chains and the amino or carboxy termini). Such substitutions, deletions or additions may involve one or more amino acids or in the case of polynucleotide, one or more nucleotide. The substitutions are preferably conservative, i.e., an amino acid is replaced by another amino acid having similar physico-chemical properties (size, hydrophobicity, charge/polarity, etc.) as well known by those of ordinary skill in the art. Functional fragments of the soluble NRP1 include a fragment or a portion of a soluble NRP1 polypeptide (e.g., the a1 and/or a2 domain(s)) or a fragment or a portion of a homologue or allelic variant of NRP1 which retains inhibiting activity, i.e., binds to SEMA3A, VEGF and/or TGF-β and inhibits the transduction of NRP1-mediated cellular activity. Non-limiting examples of NRP1-mediated cellular activity include i) vascular hyperpermeability; ii) MPs activation and recruitment; iii) inducement of apoptosis; iv) induction of pro-inflammatory cytokines (e.g., TNF-α, IL-1β) production and/or secretion. In an embodiment, the NRP1 polypeptide is at least 80, 85, 88, 90, 95, 98 or 99% identical to the polypeptide sequence of FIG. 22 (NRP1 isoform 2, SEQ ID NO: 65) or amino acids 1-859 or 22-859 of the NRP1 isoform 1 set forth in FIG. 26 (SEQ ID Nos: 66 and 69). In an embodiment, the NRP1 functional fragment comprises subdomains a1, a2, b1, b2 and/c which are/is is at least 80, 85, 88, 90, 95, 98 or 99% identical to subdomain(s) a1 (e.g., SEQ ID NO: 71 or amino acids 22-148 of SEQ ID NO: 66), a2 (e.g., SEQ ID NO: 72, or amino acids 149-275 of SEQ ID NO: 66), b1 (e.g., SEQ ID NO:73 or amino acids), b2 (e.g., SEQ ID NO: 74 or amino acids 429-589 of SEQ ID NO:66) and/or c (e.g., SEQ ID NO: 75 or amino acids 590-859 of SEQ ID NO: 66) of NRP1 as depicted in FIG. 22 or 26 (SEQ ID NOs:65 and 66 respectively). In an embodiment, the NRP1 is a functional variant which includes variations (conservative or non conservative substitution(s) and/or deletion(s)) in amino acids which are not conserved between rat, mouse and human NRP1 (see FIG. 26 and consensus sequence set forth in SEQ ID NO: 69). Preferably, the NRP1 polypeptide/polynucleotide or fragment thereof is human.

TABLE 2 Non-limiting examples of substitutions in the soluble NRP1 polypeptide/NRP1 traps of the present invention. WT Amino acid (with ref. to FIG. 26, Exemplary SEQ ID NO: 66) Domain substitution(s) N24 a1 Serine E26 a1 Lysine D29 a1 Glycine S35 a1 Asparagine D62 a1 Glutamic acid M68 a1 Isoleucine F90 a1 Isoleucine N96 a1 Glycine H98 a1 Arginine F99 a1 Leucine R100 a1 Tryptophan P110 a1 Serine T153 a2 Alanine S155 a2 Threonine S170 a2 Cysteine V177 a2 Isoleucine P196 a2 Glutamine D219 a2 Glutamic acid I242 a2 Valine 269 a2 Isoleucine 298 b1 Glycine A303 b1 valine N323 b1 Lysine K359 b1 Arginine I360 b1 Valine V362 b1 Isoleucine T371 b1 Serine I372 b1 Leucine P378 b1 Alanine V379 b1 Isoleucine L380 b1 Isoleucine V392 b1 Phenylalanine, leucine A393 b1 Glycine P396 b1 Proline, serine A409 b1 Valine T410 b1 Serine T410 b1 Serine S449 b2 Alanine G453 b2 Alanine S469 b2 Threonine A476 b2 Serine S479 b2 Proline I481 b2 Threonine I487 b2 Valine E491 b2 Aspartic acid 498 b2 Valine G518 b2 Alanine M528 b2 Threonine A553 b2 Alanine P555 b2 Serine, threonine A556 b2 Proline G572 b2 Serine A587 c Valine L599 c Proline D601 c Histidine V634 c Isoleucine N667 c Serine 669 c Alanine K672 c Arginine S674 c Arginine N717 c Serine R741 c Histidine A755 c Valine I756 c Valine S805 c Proline A813 c Threonine P820 c Threonine G835 c deletion E838 c Lysine E854 c Aspartic acid T410 b1 Serine S449 b2 Alanine

Antibodies

NRP1 cellular activity can be inhibited by using an agent which blocks NRP1 binding to one or more of its ligands (e.g., SEMA3A, VEGF and/or TGF-β). One example of such agent is an antibody which binds to NRP1 and blocks the binding of NRP1 to SEMA3A, VEGF and/or TGF-β.

Alternatively, inhibition of NRP1-mediated cellular signaling can be achieved by using an agent which blocks the binding of an NRP1 ligand to the NRP1 polypeptide. Non-limiting examples of such agent includes an antibody which binds to SEMA3A, VEGF or TGF-β and blocks their respective binding to NRP1.

In a particular aspect of the present invention, antibodies targeting NRP1 block SEMA3A binding to the receptor but do not substantially interfere with VEGF and/or TGF-β binding to NRP1. In an embodiment, the anti NRP1 antibody binds to the a1a2 domains of the NRP1 polypeptide. In another embodiment, the anti NRP1 antibody binds to subdomains a1 or a2 of the NRP1 polypeptide.

As noted above, anti SEMA3 A antibodies may be used to inhibit (i.e., reduce completely or partially) NRP1-mediated cellular signaling by blocking SEMA3A binding to NRP1. Useful anti SEMA3A antibodies bind to the SEMA domain of SEMA3A and block the interaction with NRP1. In embodiments the anti-SEMA3A antibodies used in accordance with the present invention include antibodies binding to SEMA3A polypeptide domains comprising amino acid residues 252-260, 359-366 or 363-380 of SEMA3A. SEMA3A antibodies which inhibit the binding of SEMA3A to NRP1 are known in art and may be used in accordance with the present invention.

As used herein, the expression “anti NRP1 antibody” refers to an antibody that specifically binds to (interacts with) a NRP1 protein and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the NRP1 protein. Similarly, the expression “anti SEMA3A antibody”, “anti VEGF antibody” or “anti TGF-β antibody” refers to an antibody that specifically binds to (interacts with) a SEMA3A, VEGF or TGF-β protein respectively and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the targeted SEMA3A/VEGF/TGF-β protein.

Antibodies that can be used in accordance with the present invention include polyclonal, monoclonal, humanized as well as chimeric antibodies. The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), nanobodies, shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies™, antibody-T-cell epitope fusions (Troybodies) or Peptibodies.

Anti-human NRP1/sem3A/VEGF/TGF-β antibodies have been previously prepared and are also commercially available from various sources including Santa Cruz, AbCam, and Cell Signaling.

In general, techniques for preparing antibodies (including monoclonal antibodies, hybridomas and humanized antibodies when their sequences are known) and for detecting antigens using antibodies are well known in the art and various protocols are well known and available.

Inhibition of the Expression of NRP1 or NRP1 Ligands

Various approaches are available for decreasing the expression (at the mRNA or protein level) of NRP1 or its ligands (e.g., SEMA3A, VEGF or TGF-β) to inhibit NRP1 mediated cell signaling and thus reduce inflammation and hyperactivation of innate immune response (i.e., i) production and/or secretion of pro-inflammatory cytokines; ii) recruitment of mononuclear phagocytes (MPs); iii) vascular hyperpermeabilization; and/or iv) edema, v) neuronal damage, choroidal neovascularization etc.). Non-limiting example includes the use of small hairpin shRNA (RNAi), antisense, ribozymes, TAL effectors targeting the NRP1, SEMA3A, VEGF or Tgf-β promoter or the like.

Expression in cells of shRNAs, siRNAs, antisense oligonucleotides or the like can be obtained by delivery of plasmids or through viral (e.g., lentiviral vector) or bacterial vectors.

Therefore, in alternative embodiments, the present invention provides antisense, shRNA molecules and ribozymes for exogenous administration to effect the degradation and/or inhibition of the translation of mRNA of interest. Preferably, the antisense, shRNA molecules and ribozymes target human NRP1, SEMA3A, VEGF and/or Tgf-β expression. Examples of therapeutic antisense oligonucleotide applications include: U.S. Pat. No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No. 5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617, issued Feb. 11, 1992; U.S. Pat. No. 5,166,195 issued Nov. 24, 1992; U.S. Pat. No. 5,004,810, issued Apr. 2, 1991; U.S. Pat. No. 5,194,428, issued Mar. 16, 1993; U.S. Pat. No. 4,806,463, issued Feb. 21, 1989; U.S. Pat. No. 5,286,717 issued Feb. 15, 1994; U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423; BioWorld Today, Apr. 29, 1994, p. 3.

Preferably, in antisense molecules, there is a sufficient degree of complementarity to the mRNA of interest to avoid non-specific binding of the antisense molecule to non-target sequences under conditions in which specific binding is desired, such as under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. The target mRNA for antisense binding may include not only the information to encode a protein, but also associated ribonucleotides, which for example form the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. A method of screening for antisense and ribozyme nucleic acids that may be used to provide such molecules as Shc inhibitors of the invention is disclosed in U.S. Pat. No. 5,932,435.

Antisense molecules (oligonucleotides) of the invention may include those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages, phosphorothioates and those with CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂ (known as methylene(methylimino) or MMI backbone), CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—P—O—CH₂). Oligonucleotides having morpholino backbone structures may also be used (U.S. Pat. No. 5,034,506). In alternative embodiments, antisense oligonucleotides may have a peptide nucleic acid (PNA, sometimes referred to as “protein nucleic acid”) backbone, in which the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone (Nielsen et al., 1991, Science 254:1497 and U.S. Pat. No. 5,539,082). The phosphodiester bonds may be substituted with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.

Oligonucleotides may also include species which include at least one modified nucleotide base. Thus, purines and pyrimidines other than those normally found in nature may be used. Similarly, modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected. Examples of such modifications are 2′-O-alkyl- and 2′-halogen-substituted nucleotides. Some specific examples of modifications at the 2′ position of sugar moieties which are useful in the present invention are OH, SH, SCH₃, F, OCN, O(CH₂)_(n) NH₂ or O(CH₂)_(n) CH where n is from 1 to about 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CH; OCF₃; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. One or more pentofuranosyl groups may be replaced by another sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar.

In some embodiments, the antisense oligonucleotides in accordance with this invention may comprise from about 5 to about 100 nucleotide units. As will be appreciated, a nucleotide unit is a base-sugar combination (or a combination of analogous structures) suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds forming a backbone structure.

In a further embodiment, expression of a nucleic acid encoding a polypeptide of interest (e.g., SEMA3A or NRP1), or a fragment thereof, may be inhibited or prevented using RNA interference (RNAi) technology, a type of post-transcriptional gene silencing. RNAi may be used to create a pseudo “knockout”, i.e. a system in which the expression of the product encoded by a gene or coding region of interest is reduced, resulting in an overall reduction of the activity of the encoded product in a system. As such, RNAi may be performed to target a nucleic acid of interest or fragment or variant thereof, to in turn reduce its expression and the level of activity of the product which it encodes. Such a system may be used for functional studies of the product, as well as to treat disorders related to the activity of such a product. RNAi is described in for example published US patent applications 20020173478 (Gewirtz; published Nov. 21, 2002) and 20020132788 (Lewis et al.; published Nov. 7, 2002). Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, Tex., USA) and New England Biolabs Inc. (Beverly, Mass., USA).

The initial agent for RNAi in some systems is a dsRNA molecule corresponding to a target nucleic acid. The dsRNA (e.g., shRNA) is then thought to be cleaved into short interfering RNAs (siRNAs) which are 21-23 nucleotides in length (19-21 bp duplexes, each with 2 nucleotide 3′ overhangs). The enzyme thought to effect this first cleavage step has been referred to as “Dicer” and is categorized as a member of the RNase III family of dsRNA-specific ribonucleases. Alternatively, RNAi may be effected via directly introducing into the cell, or generating within the cell by introducing into the cell a suitable precursor (e.g. vector encoding precursor(s), etc.) of such an siRNA or siRNA-like molecule. An siRNA may then associate with other intracellular components to form an RNA-induced silencing complex (RISC). The RISC thus formed may subsequently target a transcript of interest via base-pairing interactions between its siRNA component and the target transcript by virtue of homology, resulting in the cleavage of the target transcript approximately 12 nucleotides from the 3′ end of the siRNA. Thus the target mRNA is cleaved and the level of protein product it encodes is reduced.

RNAi may be effected by the introduction of suitable in vitro synthesized siRNA (shRNAs) or siRNA-like molecules into cells. RNAi may for example be performed using chemically-synthesized RNA. Alternatively, suitable expression vectors may be used to transcribe such RNA either in vitro or in vivo. In vitro transcription of sense and antisense strands (encoded by sequences present on the same vector or on separate vectors) may be effected using for example T7 RNA polymerase, in which case the vector may comprise a suitable coding sequence operably-linked to a T7 promoter. The in vitro-transcribed RNA may in embodiments be processed (e.g. using E. coli RNase III) in vitro to a size conducive to RNAi. The sense and antisense transcripts are combined to form an RNA duplex which is introduced into a target cell of interest. Other vectors may be used, which express small hairpin RNAs (shRNAs) which can be processed into siRNA-like molecules. Various vector-based methods and various methods for introducing such vectors into cells, either in vitro or in vivo (e.g. gene therapy) are known in the art.

Accordingly, in an embodiment expression of a nucleic acid encoding a polypeptide of interest (or a fragment thereof e.g., soluble NRP1, NRP1 derived traps, may be inhibited by introducing into or generating within a cell an siRNA or siRNA-like molecule corresponding to a nucleic acid encoding a polypeptide of interest (e.g. SEMA3A or NRP1), or a fragment thereof, or to an nucleic acid homologous thereto. “siRNA-like molecule” refers to a nucleic acid molecule similar to an siRNA (e.g. in size and structure) and capable of eliciting siRNA activity, i.e. to effect the RNAi-mediated inhibition of expression. In various embodiments such a method may entail the direct administration of the siRNA or siRNA-like molecule into a cell, or use of the vector-based methods described above. In an embodiment, the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecule is about 21-23 nucleotides in length. In an embodiment, siRNA or siRNA-like molecule comprises a 19-21 bp duplex portion, each strand having a 2 nucleotide 3′ overhang. In embodiments, the siRNA or siRNA-like molecule is substantially identical to a nucleic acid encoding a polypeptide of interest, or a fragment or variant (or a fragment of a variant) thereof. Such a variant is capable of encoding a protein having activity similar to the polypeptide of interest.

A variety of viral vectors can be used to obtain shRNA/RNAi expression in cells including adeno-associated viruses (AAVs), adenoviruses, and lentiviruses. With adeno-associated viruses and adenoviruses, the genomes remain episomal. This is advantageous as insertional mutagenesis is avoided. It is disadvantageous in that the progeny of the cell will lose the virus quickly through cell division unless the cell divides very slowly. AAVs differ from adenoviruses in that the viral genes have been removed and they have diminished packing capacity. Lentiviruses integrate into sections of transcriptionally active chromatin and are thus passed on to progeny cells. With this approach there is increased risk of insertional mutagenesis; however, the risk can be reduced by using an integrase-deficient lentivirus.

Pharmaceutical Compositions and Kits

Agents which inhibit NRP1-dependent cell signaling (i.e., NRP1 inhibitors) of the present invention can be administered to a human subject by themselves or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s) at doses to treat or prevent the targeted disease or condition or to raise the desired cellular response.

Mixtures of these compounds (e.g., NRP1 trap, antibodies, dominant negative, small inhibitory peptides or the like) can also be administered to the subject as a simple mixture or in suitable formulated pharmaceutical compositions. A therapeutically effective dose further refers to that amount of the compound or compounds sufficient to result in the prevention or treatment of the targeted inflammatory disease or condition (e.g., such as septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis and neuroinflammatory conditions such as diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease) or to provide the desired cellular or physiological response (e.g., amount sufficient to i) reduce edema, ii) reduce activation/recruitment of mononuclear phagocytes (e.g., microglia or macrophages), iii) reduce production or secretion of inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6, etc.); iv) reduce pathological neovascularization; v) reduce vascular degeneration, etc.,).

As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, physiological media, and the like that are physiologically compatible. In embodiments the carrier is suitable for ocular administration. In other embodiments the carrier is suitable for systemic administration. In other embodiments the carrier is suitable for oral administration.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents, such as for ocular, systemic or oral application, is well known in the art. Except insofar as any conventional media or agent is incompatible with the compounds of the invention, use thereof in the compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

The present invention also concerns kits or commercial packages for use in the methods of the present invention. Such kits may comprises compounds of the present invention (e.g., compounds which inhibit NRP1 cell signaling, including SEMA3A-mediated cell signaling such as traps, antibodies, shRNA, cells, vectors, nucleic acids) optionally with instructions to use the kit.

Routes of Administration/Formulations

Suitable routes of administration may, for example, include systemic, oral and ocular (eye drops or intraocular injections). Preferred routes of administration comprise eye drops and intraocular injections for eye conditions, oral for chronic inflammatory conditions and systemic for sepsis and certain neuronal conditions such as stroke. The formulations may also be in the form of sustained release formulations.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.

The compounds may be formulated for ocular administration e.g., eye drops or ocular injections. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a cell-specific antibody or other delivery system (e.g., to target for example a specific tissue (e.g., brain) or cell type (e.g., microglia or macrophages)). Nanosystems and emulsions are additional well known examples of delivery vehicles or carriers for drugs. Another example is the Encapsulated Cell Therapy (ECT) delivery system from Neurotech's, for eye diseases. ECT is a genetically engineered ocular implant that enables continuous production of therapeutic proteins to the eye for over 2 years. Additionally, the therapy is reversible by simply removing the implant. The ECT implant is inserted into the vitreous through a single incision and sutured in place in a 20-minute outpatient surgical procedure.

Effective Dosage

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art.

The effective dose of the compound inhibits the cellular signaling function of NRP1 sufficiently to reduce or prevent one or more physiological or cellular responses (e.g., vascular hyperpermeability, blood retinal barrier leakage, edema, MPs activation and/or recruitment, proinflammatory cytokines production and/or secretion, neovascularization, neuronal damage, etc.) or to prevent or treat a given inflammatory disease or condition, without causing significant adverse effects. Certain compounds which have such activity can be identified by in vitro assays that determine the dose-dependent inhibition of NRP1-mediated cell signaling inhibitors (e.g., agents which directly target the expression or activity of NRP1 or agents which targets the expression or activity (e.g., binding) of ligands of NRP1.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cellular assays. For example, a dose can be formulated in cellular and animal models to achieve a circulating concentration range that includes the IC₅₀ as determined in cellular assays (i e., the concentration of the test compound which achieves a half-maximal inhibition of the cellular signaling function of NRP1, usually in response to inflammatory mediators such as Il-1β or other activating stimulus such as hypoxia, ischemia, cellular stress, ER stress, etc.

A therapeutically effective amount refers to that amount of the compound that results in amelioration of symptoms in a subject. Similarly, a prophylactically effective amount refers to the amount necessary to prevent or delay symptoms in a patient (e.g., NRP1-mediated vascular hyperpermeability, spotted and/or blurry vision, pericytes loss, macular edema, retinal swelling, blood retinal barrier leakage, mononuclear phagocytes recruitment, production and secretion of pro-inflammatory cytokines, vascular degeneration, pathological neovascularization, neuronal damage, etc.). Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the maximum tolerated dose (MTD) and the ED (effective dose for 50% maximal response). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between MTD and ED50. Compounds which exhibit high therapeutic indices are preferred. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

Dosage amount and interval may be adjusted individually to provide levels of the active compound which are sufficient to maintain the NRP1 modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e. g. the concentration necessary to achieve substantial inhibition of SEMA3A expression or activity (e.g., binding to NRP1 receptor) Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

Definitions

For clarity, definitions of the following terms in the context of the present invention are provided.

As used herein, the term “Neuropilin-1 receptor” or “NRP1” receptor refers to neuropilin-1 and its isoforms, and allelic/polymorphic forms (e.g., HGNC: 8004; Entrez Gene: 8829; Ensembl: ENSG00000099250; OMIM: 602069; and UniProtKB: 014786; GenBank Acc. No. AAH07737.1, FIG. 22, SEQ ID NO: 65). NRP1 is a non-tyrosine kinase multifunctional receptor having the particular ability to bind three structurally dissimilar ligands via distinct sites on its extracellular domain. It binds SEMA3A^(18,19) (for example provoking cytoskeletal collapse) and VEGF₁₆₅, enhancing binding to VEGFR2 (for example increasing its angiogenic potential). It also binds to TGF-β. Moreover, genetic studies show that NRP1 distinctly regulates the effects of VEGF and SEMA3A on neuronal and vascular development. Hence, depending on the ligand, NRP1-mediated cellular response varies.

The basic structure of neuropilin-1 comprises 5 domains: Three extracellular domains (a1a2 (CUB), b1b2 (FV/FVIII) and c (MAM)), a transmembrane domain and a cytoplasmic domain (See FIGS. 19A and 22 and SEQ ID NO: 65 and 66 and 68). The a1a2 domain is homologous to complement components C1r and C1s (CUB) which generally contain 4 cysteine residues forming disulfide bridges. This domain binds SEMA3A. Domains b1b2 (FV/FVIII) binds to VEGF. Amino acid Y297 in subdomain b1 is important for binding to VEGF as substitution of Y297 to an alanine significantly reduces VEGF binding to NRP1. There exists several splice variants isoforms and soluble forms of NRP1 which are all encompassed by the present invention.

“Homology” and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid/polynucleotide sequence is “homologous” to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term ‘homologous’ does not infer evolutionary relatedness). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical. As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than about 25% identity, with any of the nucleic acids and polypeptides disclosed herein.

Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridisation to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. For example, in an embodiment, the compound of the present invention is an antisense/RNAi or shRNA that hybridizes to an NRP1 or SEMA3A nucleic acid sequence (preferably a human sequence).

As used herein the term “treating” or “treatment” in reference to inflammatory diseases or conditions (e.g., retinopathies, cerebral ischemia, stroke, sepsis, etc.) is meant to refer to a reduction/improvement in one or more symptoms or pathological physiological responses associated with said disease or condition. Non-limiting examples include edema, swelling, itching, pain, vascular hyperpermeability; blood retinal barrier integrity, increase in SEMA3A, VEGF and/or TGF-beta expression, mononuclear phagocyte recruitment/chemotaxis, production and/or secretion of proinflammatory cytokines, vascular or neuronal degeneration, etc.

As used herein the term “preventing” or “prevention” in reference to inflammatory diseases or conditions is meant to refer to a reduction in the progression or a delayed onset of at least one symptom associated with the disease or condition.

The articles “a,” “an” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

The term “including” and “comprising” are used herein to mean, and re used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”.

The terms “such as” are used herein to mean, and is used interchangeably with, the phrase “such as but not limited to”.

The present invention is illustrated in further details by the following non-limiting examples.

Example 1 Materials and Methods (Examples 2-9 and 12)

Generation of LyzM-cre/Nrpfl/fl mice. C57131/6 wild-type (WT) were purchased from The Jackson Laboratory. LyzM-Cre (Lyz2tm1(cre)Ifo/J; no. 004781) and NRP1 floxed mice (Nrp1tm2Ddg/J; no. 005247) were purchased from The Jackson Laboratory and bread to obtain LyzM-cre/Nrpfl/fl with NRP1-deficient myeloid cells.

O₂-induced retinopathy. Mouse pups (WT or LyzM-Cre (Jackson Laboratory) or LysM-Cre/Nrp1fl/fl) and their fostering mothers (CD1, Charles River) were exposed to 75% O₂ from postnatal day 7 (P7) to day 12 and returned to room air (52). This model serves as a proxy to human ocular neovascular diseases such as diabetic retinopathy characterized by a late phase of destructive pathological angiogenesis (53, 54). Upon return to room air, hypoxia-driven neovascularization (NV) develops from P14 onwards (26). Eyes were enucleated at different time points and the retinas dissected for FACS analysis or mRNA analysis as described. In other experiments, dissected retinas were flatmounted and incubated overnight with fluoresceinated isolectin B4 (1:100) in 1 mM CaCl₂ to determine extent of avascular area or neovascularization area at P17 using ImageJ and the SWIFT-NV method (55).

FACS of digested retinas and spleen. Retinas from WT or LysM-Cre/Nrp1fl/fl mice were homogenized and incubated in a solution of 750 U/mL DNaseI (Sigma) and 0.5 mg/mL of collagenase D (Roche) for 15 min at 37° C. with gentle shaking. Homogenates were then filtered with a 70 μm cell strainer and washed in PBS+3% fetal bovine serum. Spleen samples were homogenized and incubated with 1 mg/mL of collagenase D for 10 min at 37° C. Homogenates were washed in PBS+3% fetal bovine serum and the pellets were resuspended and incubated in lysis buffer (10 mM KCHO₃; 150 mM NH₄Cl; 0.1 mM EDTA) for 5 min at room temperature. Cell suspensions (retina or spleen) were incubated with LEAF™ purified anti-mouse CD16/32 (Biolegend) for 15 min at room temperature to block Fc receptors. Cells were then incubated for 30 min at room temperature with the following antibodies: FITC anti-mouse/human CD11b (Biolegend), PE/CY7 anti-mouse Ly-6G/Ly-6C (Gr-1; Biolegend), Pacific Blue™ anti-mouse F4/80 (Biolegend), 7AAD (BD Biosciences) and anti-mNeuropilin-1 Allophycocyanin conjugated Rat IgG2A (R&D Systems) or Rat IgG2A Isotype Control Allophycocyanin conjugated (R&D Systems).

For analysis of CX3CR1 and CD45 expression, additional extracellular staining was performed using the above mentioned antibodies supplemented with Alexa Fluor 700 anti-mouse CD45.2 (Biolegend) and anti-mouse CX3CR1 Phycoerythrin conjugated Goat IgG (R&D Systems) or Goat IgG Isotype. Control Phycoerythrin conjugated FACS was performed on a LSRII (BD Biosciences) device and data were analysed using FlowJo™ software (version 7.6.5).

BrdU injections. Wild-type mice subjected to OIR or kept in normoxic conditions were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU; Sigma) at the dose of 1 mg/mouse dissolved in PBS at P13.

Analysis of BrdU incorporation. The staining was performed on the retinal cells from P14 WT mice. Samples were obtained as described above. Extracellular staining was performed as described above (CD45.2 (intermediate/low); Gr-1−; D11b+, F4/80+; 7AAD). Cells were then fixed with Cytofix/Cytoperm™ Buffer (BD Biosciences) for 30 min and permeabilised with Perm/Wash™ Buffer (BD Biosciences) for 10 min. Next, cells were treated with 300 ug/mL of DNAse for 1 h at 37° C. and washed with Perm/Wash™. Intracellular staining of BrdU was performed using anti-BrdU-PE antibodies (Ebioscience) or mouse IgG1 κ Isotype Control PE conjugated (Ebioscience) for 25 min at 4° C. Cells were then washed in Perm/Wash™ and resuspended in PBS-F3% fetal bovine serum before FACS analysis on a LSRII (BD Biosciences).

Vitrectomy. All patients previously diagnosed with PDR were followed and operated by a single vitreoretinal surgeon (FAR). Control patients were undergoing surgical treatment for non-vascular pathology (ERM (epiretinal membrane) or MH (macular hole)) by the same surgeon. In an operating room setting, patients underwent surgery under local retro/peribulbar anesthesia. A 5% povidone-iodine solution was used to clean the periocular skin and topical instillation into the eye and within the cul-de-sac was left in place for 5 minutes. Three-port 25-gauge transconjunctival pars plana vitrectomy was performed through 25-gauge valved cannulas (Alcon). Under microscope visualization using a wide-angle viewing system (Resight™, Zeiss), undiluted vitreous was collected with a 25-gauge vitrector. After vitreous biopsy, the infusion line was opened and vitrectomy and membrane peeling was performed in the usual fashion to treat diabetic vitreous hemorrhage and tractional retinal detachment. This was followed by panretinal endolaser photocoagulation, fluid-air exchange, and intravitreal anti-VEGF injection.

Quantification of SEMA3A protein by ELISA. Vitreous samples were frozen on dry ice and immediately after biopsy and stored at −80°. Samples were centrifuged at 15000×g for 5 minutes at 4° C. prior to analysis. SEMA3A levels were quantified in supernatants using enzyme-linked immunosorbent assays (ELISAs) following manufacturer's instructions (USCN Life Science Inc.).

Assessment of SEMA3A protein levels by Western-blot. Equal volumes of vitreous fluid (20 uL) from PDR and control patients were assessed by standard SDS-PAGE technique for the presence of SEMA3A (Abcam).

Real-time PCR analysis. RNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) and digested with DNase I to prevent amplification of genomic DNA. Reversed transcription was performed using M-MLV reverse transcriptase (Life Technologies) and gene expression analyzed using Sybr™ Green (BioRad) in an ABI Biosystems Real-Time PCR machine. β-actin was used as a reference gene (see Table 2 in Example 10 for details on the sequence of the oligonucleotides used.

Immunohistochemistry. For visualization of pan-retinal vasculature, flatmount retinas were stained with stained with Rhodamine labeled Griffonia (Bandeiraea) Simplicifolia Lectin I (Vector Laboratories, Inc.) in 1 mM CaCl₂ in PBS for retinal vasculature and anti-rat Neuropilin-1 antibody, (goat IgG; R&D Systems) and IBA1 (rabbit polyclonal; Wako).

Primary peritoneal macrophages culture. Adult WT or LyzMcre/NRP1fl/fl mice were anesthetized with 2% isoflurane in oxygen 2 L/min and then euthanized by cervical dislocation. Then, a small incision in abdominal skin of mouse was performed. Skin was pulled to each size of the mouse and peritoneal cavity was washed with 5 ml of PBS plus 3% FBS for 2 min. Then, the harvested cells were centrifuged for 5 min at 1000 rpm, resuspended in medium (DMEM F12 plus 10% FBS and 1% Streptomycin/Penicillin) and plated. After 1 h of culture at 37° C. under a 5% CO₂ atmosphere the medium was changed and cells were cultured for the next 24 h in the same conditions before use in cytokine or transwell migration assay.

Transwell migration assay. Migration assays were performed in 24-well plates with 8 μm pore inserts. Primary peritoneal macrophages (5×105 cells) resuspended in 200 μl of medium (DMEM F12 plus 10% FBS and 1% Streptomycin/Penicillin) were added to the upper chamber. 800 μl of medium with or without migratory factors: MCP-1 (100 ng/ml), SEMA3A (100 ng/ml), and VEGF₁₆₅ (50 ng/ml) was added to the lower chamber. Cells were allowed to migrate through the insert membrane overnight at 37° C. under a 5% CO₂ atmosphere. In some experiments, cells were first pretreated with Y-27632 (Sigma), selective ROCK (Rho-associated coiled coil forming protein serine/threonine kinase) inhibitor (100 μg/ml) for 1 h at 37° C. The inserts were then washed with PBS, and nonmigrating cells were swabbed from the upper surface of the insert membrane with cotton buds. Then the membranes with migrated cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes, washed twice with PBS and mounted on the slide. The cells were stained using mounting medium with DAPI (Vector Laboratories, Inc.). Then, 9 random fields per each membrane were photographed using an inverted fluorescence microscope at 20× magnification and the cells were counted using ImageJ software.

Choroidal explants and microvascular sprouting assay. The ex vivo choroid explants and quantification of microvascular sprouting as described previously(56). Briefly, choroids from LysM-Cre/Nrp1^(+/+) and LysM-Cre/Nrp1^(fl/fl) mice (n=6 for each condition) were dissected shortly after enucleating eyes. After plating segmented choroids into 24 well tissue culture plates and covering with Matrigel™ (BD Biosciences), samples were treated with either EGM™-2 medium, EGM-2 medium with PBS filled liposome (liposome-PBS), or EGM™-2 medium with Dichloromethylenediphosphonic acid disodium salt filled liposome (liposome-Clodronate) (Sigma). The packaging of liposomes was performed according to (57). Twelve hours later, liposomes containing passenger compounds were removed from the wells followed by washing with PBS. Macrophages from primary peritoneal macrophage cultures (from either LysM-Cre/Nrp1^(+/+) or LysM-Cre/Nrp1^(fl/fl) mice) were added to choroidal explant cultures to investigate the impact of macrophages on microvascular sprouting.

Soluble recombinant NRP1. Wild-type mice subjected to OIR were intravitreally injected with rmNRP1 trap-1 (FIGS. 19C and 20R, SEQ ID NO: 25) from plasmid (29) or R&D Systems at P12.

Recombinant proteins used. Recombinant mouse CCL2/JE/MCP-1 (from E. coli) (R&D Systems) concentration used in vitro 100 ng/ml. Recombinant human SEMA3A Fc chimera (from murine myeloma cell line, NS0) (R&D Systems) concentration used in vitro 100 ng/ml. —Recombinant human VEGF₁₆₅ (PeproTech) concentration used in vitro 50 ng/ml.

Statistical analyses. Data are presented as mean±s.e.m. Student's T-test and ANOVA were used, where appropriate, to compare the different groups; a P<0.05 was considered statistically different. For ELISA, statistical analysis was performed using nonparametric Mann-Whitney test (GraphPad Prism).

Study approval: Human samples. We obtained approval of human clinical protocol and informed consent form by Maisonneuve-Rosemont Hospital (HMR) ethics committee (Ref. CER: 10059) and recruitment of patients for local core vitreal biopsy sampling from patients afflicted with T1DM or T2DM. The entire procedure was performed as an outpatient procedure in the minor procedure room within the ambulatory clinic from the Department of Ophthalmology at Maisonneuve-Rosemont Hospital. All instruments were opened and handled in a sterile manner. The study conforms to the tenets of the declaration Helsinki.

Study approval: Animals. All studies were performed according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of the University of Montreal in agreement with the guidelines established by the Canadian Council on Animal Care. C57131/6 wild-type (WT) were purchased from The Jackson Laboratory. LyzM-Cre (Lyz2tm1(cre)Ifo/J; no. 004781) and Neuropilin 1 floxed mice (Nrp1tm2Ddg/J; no. 005247) were purchased from The Jackson Laboratory.

TABLE 3 Characteristics of Vitrectomy Patients Db Duration Sample Age type (years) Retinopathy Analysis C1 74 na na MH WB/ELISA C2 54 na na MMD WB/ELISA C3 72 na na ERM WB/ELISA C4 77 na na ERM WB/ELISA C5 82 na na MH WB/ELISA C6 62 na na ERM ELISA C7 65 na na MH ELISA C8 69 na na ERM ELISA C9 75 na na MH/Cataract ELISA C10 77 na na Ret. Det. ELISA C11 69 na na ERM ELISA C12 68 na na ERM ELISA C13 81 na na ERM ELISA C14 70 na na ERM ELISA C15 65 na na MH ELISA C16 74 na na MH ELISA C17 75 na na MH ELISA PDR1 62 2 13 PDR WB/ELISA PDR2 79 2 33 PDR WB/ELISA PDR3 73 2 15 PDR WB/ELISA PDR4 74 2 10 PDR WB/ELISA PDR5 54 1 20 PDR WB/ELISA PDR6 60 2 34 PDR WB/ELISA PDR7 77 2 34 PDR WB/ELISA PDR8 71 2 10 PDR ELISA PDR9 35 — — PDR ELISA PDR10 69 2 40 PDR ELISA PDR11 78 —  5 PDR ELISA PDR12 36 2 — PDR ELISA PDR13 81 1 30 PDR ELISA PDR14 70 2 30 PDR ELISA PDR15 74 — 35 PDR ELISA PDR16 67 2 30 PDR ELISA PDR17 69 2  2 PDR ELISA MH: Macular hole MMD: Myopic Macular Degeneration ERM: Epiretinal Membrane PDR: Proliferative Diabetic Retinopathy Ret. Det.: Retinal Detachement

Example 2 NRP1 Identifies a Population of Mononuclear Phagocytes (MPs) that are Mobilized Secondary to Vascular Injury

To determine whether MPs (mononuclear phagocytes) such as microglia or macrophages partake in the vascular pathogenesis associated with proliferative retinopathies, FACS analysis was first carried-out on whole mouse retinas to elucidate the kinetics of macrophage/microglial accumulation throughout the evolution of oxygen-induced retinopathy (OIR, FIG. 1A, 75% oxygen from P7-P12 (postnatal day 7-12) to induce vasoobliteration and room air until P17 to attain maximal pre-retinal neovascularization (26,33)) (FIG. 1B,E,H). Results revealed significantly higher numbers of retinal macrophage/microglial cells (Gr-1−, F4/80+, CD11b+, cells, data not shown) in OIR at all time points analysed including a 36% increase during the vaso-obliterative phase at P10 (P=0.0004) (FIG. 1C), a 63% rise during the neovascular phase at P14 (P<0.0001) (FIG. 1F) and a 172% surge during maximal neovascularization at P17 (p=0.0006) (FIG. 11).

Importantly, at each time point investigated, we observed a proportional increase in NRP1-positive MPs in OIR with a rise of 37% at P10 (P=0.0240) (FIG. 1D), 61% at P14 (P=0.0196) (FIG. 1G) and 155% at P17 (P=0.0058) (FIG. 1J) suggesting that this subpopulation of NRP1-positive MPs was being recruited to the neuroretina during the progression of the disease. For all OIR experiments, weights of mouse pups were recorded (data not shown) to ascertain adequate metabolic health (35).

In order to establish the role of MP-resident NRP1 in retinopathy, a myeloid specific knockout of NRP1 was generated by intercrossing Nrp1 floxed mice with LysM-Cre mice(36) yielding LysM-Cre/Nrp1^(fl/fl) progeny. The resulting mice showed an ˜80% decrease in NRP1 expression in retinal MPs when compared to LysM-Cre/Nrp1^(+/+) littermate controls (P=0.0004) (FIG. 1K). Of note, mice tested negative for the rd8 mutation of the Crb1 gene (37). LysM-Cre/Nrp1^(fl/fl) mice did not show any difference in body weight, size, or open-field activity when compared with littermates throughout the period of experimentation (from P1-P17) (data not shown) and had similar numbers of resident retinal microglia (data not shown). Remarkably, deletion of NRP1 on myeloid cells fully abrogated the entry of macrophages/microglia at P10 and P14 OIR (FIG. 1L-O) revealing the critical role for this receptor in MP chemotaxis during the early stages of ischemic retinal injury. At P17, following maximal pathological neovascularization, MP infiltration occurs largely independent of NRP1 (FIGS. 1P, Q and R). Consistent with a potential microglial identity, the NRP1-expressing Gr1−/CD11b+/F4/80+ cells identified above express high levels of CX3CR1 and intermediate/low levels of CD45 (FIG. 1S and data not shown). As expected, in LysM-Cre/Nrp1^(fl/fl) retinas, CD45low/CX3CR1 high MPs are devoid of NRP1 (FIG. 1T).

Example 3 NRP1⁺ Myeloid Cells Localize to Sites of Pathological Neovascularization in the Retina

Given the pronounced influx of NRP1⁺ macrophage/microglia during OIR, the localization of these cells during the progression of disease was next determined. Immunofluorescence on retinal flatmounts revealed that NRP1-positive macrophage/microglia (co-labelled with IBA1 and NRP1) were intimately associated with nascent pathological tufts at P14 of OIR (FIG. 2A-C) as well as mature tufts at P17 of OIR (FIG. 2D-F). White arrows in FIGS. 2B and 2E point to NRP1-positive MPs associated with pre-retinal tufts. NRP1 was also expressed by endothelial cell on the endothelium of neovascular tufts as previously reported (21). Consistent with data presented in FIG. 1, LysM-Cre/Nrp1fl/fl mice had lower numbers of macrophage/microglia and less pronounced neovascularization (see below for full quantification) (FIG. 2 G-K).

Example 4 SEMA3A is Elevated in the Vitreous of Patients Suffering from Active Proliferative Diabetic Retinopathy

To establish the clinical relevance of our findings on the obligate role of NRP1 in MP chemotaxis in retinopathy, the concentrations of SEMA3A directly in the vitreous of patients suffering from active PDR was determined. Seventeen samples of undiluted vitreous were obtained from patients suffering from PDR and 17 from control patients with nonvascular pathology. Detailed characteristics of patients are included in Table 1 (Example 1). Control patients (20) presented with non-vascular pathology and showed signs of non-diabetes-related retinal damage such as tractional tension on vasculature (FIG. 3A,B (white arrow)) secondary to fibrotic tissue and macular bulging (FIG. 3C). In contrast, all retinas from PDR patients showed signs of disc (FIG. 3D) or pre-retinal neovascularization (FIG. 3F), with highly permeable microvessels (leakage of fluorescent dye) (FIG. 3D,G insets), microaneurisms (FIG. 3D-G) and fibrous scar tissue, indicative of advanced retinopathy (FIG. 3G). In addition, patients showed some evidence of macular edema due to compromised vascular barrier function, including cystoid formation (white arrowhead) due to focal coalescence of extravasated fluid (FIG. 3H).

Consistent with a role in PDR, ELISA-based detection of SEMA3A revealed a 5-fold higher concentrations of the protein in the vitreous humor of patients with PDR when compared to vitreous from control patients (P=0.0132) (FIG. 3I). Results were confirmed by Western blot analysis on equal volumes of vitreous where SEMA3A (125 and 95 kDa isoforms)(38, 39) were elevated in patients with PDR (FIG. 3J). Thus, upregulation of SEMA3A in the vitreous is induced in diabetic ocular pathology.

Example 5 NRP1 Ligands are Induced in the Retinal Ganglion Cell Layer During OIR

To obtain an accurate kinetic profile of expression of the two prominent ligands of NRP1 in proliferative retinopathy, levels of SEMA3A and VEGF messages in the mouse model of OIR were determined. Real-time quantitative PCR (RT-qPCR) on whole retinas revealed that SEMA3A was robustly induced in OIR both during the hyperoxic (vasodegenerative) phase at P10 and the ischemic/neovascular stage from P12 to P17 (FIG. 4A). The observed induction occurred in both wild-type and LysM-Cre/Nrp1fl/fl retinas. Conversely, as expected, VEGF transcripts rose exclusively in the ischemic phase of OIR from P12 to P17 (FIG. 4B). Importantly, VEGF was significantly less induced in LysM-Cre/Nrp1fl/fl when compared to wild-type retinas (minimally increased at P12 (P=0.0451) and ˜55% lower at P14 when compared wild-type OIR (P=0.0003)) (FIG. 4B) indicative of a healthier retina.

Next, laser capture micro-dissection (LCM) followed by RT-qPCR was performed on retinal layers in avascular zones to pinpoint the source of SEMA3A and VEGF messages in OIR (FIG. 4C). Both SEMA3A and VEGF where robustly induced in the ganglion cell layer with VEGF also increasing in the inner nuclear layer (FIG. 4D, E). Thus, the source of both ligands is geographically consistent with the localization of retinal MPs (FIG. 2).

Example 6 Mononuclear Phagocytes (MPs) do not Proliferate in the Retina after Vascular Injury

In order to determine if the noted rise in NRP1+ MPs was due to an influx from systemic circulation or an increase in MP proliferation within the retina, the local retinal proliferation of these cells was investigated. Mice were systemically injected with BrdU at P13 (24 hours prior to sacrifice) and FACS analysis was carried out on retinas (FIG. 5A) and spleens (FIG. 5B). Within the retina, Gr1−/CD11b+/F4/80+ MPs did not show significant proliferation (P=0.4708). Considerably more proliferation was observed in spleens. No significant difference was observed between Normoxia and OIR (FIG. 5C). The lack of proliferation of MPs in the retina suggest that noted accretion NRP1+ MPs during retinopathy has a systemic origin.

Example 7 SEMA3A and VEGF₁₆₅ Mobilize MPs Via NRP1

In light of the requirement of NRP1 for myeloid cell mobilization to sites of vascular lesion (FIG. 1) as well as the induction of the principal ligands of NRP1 in retinopathy (FIG. 3-4) and the likely systemic origin of these cells (FIG. 5), the propensity of these cues to provoke chemotaxis of MPs was determined. Primary macrophage cultures were isolated from wild-type mice and subjected to a Transwell Boyden chamber migration assay. Both SEMA3A (100 ng/ml) (P<0.0001) and VEGF₁₆₅ (50 ng/ml) (P=0.0027) provoked macrophage chemotaxis to similar magnitudes as positive control MCP-1 (100 ng/ml) (P<0.0001) (FIGS. 6A, B). These data were validated by demonstrating that Y-27632, a selective inhibitor ROCK (Rho-associated coiled coil forming protein serine/threonine kinase) abolished their chemotactic properties. ROCK is downstream of NRP1 signaling (40) and is known to mediate monocyte migration (41). VEGF migration was partially yet not significantly diminished suggesting a contribution from alternate receptors such as VEGFR1 as recently reported (33). Consistent with a role for NRP1 in SEMA3A and VEGF-mediated chemotaxis, macrophages from LysM-Cre/Nrp1^(fl/fl) mice were uniquely responsive to MCP-1 and not mobilized by SEMA3A or VEGF (FIG. 6C).

Example 8 NRP1+ Macrophages Potentiate Microvascular Sprouting Ex Vivo

To investigate the impact of NRP1 expressing macrophages on microvascular angiogenesis, choroid tissue from either LysM-Cre/Nrp1^(+/+) mice or LysM-Cre/Nrp1^(fl/fl) mice was isolated and grew in Matrigel™ to assess microvascular sprouting. Choroids from LysM-Cre/Nrp1^(fl/fl) mice sprout ˜20% less microvessels when compared to ones from LysM-Cre/Nrp1^(+/+) mice (p=0.018) (FIG. 7A). To investigate the role of NRP1⁺ macrophages in promoting microvascular sprouting, clodronate-liposomes were used to eliminate endogenous macrophages from the isolated choroid tissues. In explants from both LysM-Cre/Nrp1^(fl/fl) and LysM-Cre/Nrp1^(+/+) mice, PBS containing liposomes (i.e. vehicle control) had no impact on vascular sprouting, but clodronate-liposomes reduced microvascular sprouting by ˜60% (P=0.0114 for LysM-Cre/Nrp1^(+/+) choroid and P=0.0007 for LysM-Cre/Nrp1^(fl/fl) choroid) (FIGS. 7B-E). To verify whether NRP1±macrophages have a propensity to promote angiogenesis, peritoneal macrophages were extracted from LysM-Cre/Nrp1^(+/+) or LysM-Cre/Nrp1^(fl/fl) mice, and introduced into choroid explant cultures that had previously been treated with clodronate liposomes and washed. LysM-Cre/Nrp1^(+/+) macrophages robustly potentiated microvascular sprouting by 50-100% when compared to macrophages from LysM-Cre/Nrp1^(fl/fl) mice (P=0.0068 for LysM-Cre/Nrp1^(+/+) choroid and P=0.0491 for LysM-Cre/Nrp1^(fl/fl) choroid) (FIGS. 7D and E) and independent of the genotype of the choroidal explant.

Example 9 Deficiency in Myeloid-Resident NRP1 Reduces Vascular Degeneration and Pathological Neovascularization in Retinopathy

Given the obligate role of NRP1 cell signaling in MP infiltration during the early stages of OIR (FIG. 1), the impact of myeloid cell-specific ablation of NRP1 on the progression of disease was next determined. Upon exit from 75% 02 at P12, LysM-Cre/Nrp1^(fl/fl) mice showed significantly lower levels of retinal vasoobliteration when compared to wild-type (P=0.0011) and LysM-Cre/Nrp1+I+ (P<0.0001) controls (FIGS. 8A, B). This may be attributed to lower levels of IL-1β present in the retinas of LysM-Cre/Nrp1fl/fl mice (Data not shown). Importantly, at P17 when pathological neovascularization peaks (26), deletion of myeloid-resident NRP1 profoundly reduced avascular areas (˜35% when compared to wild-type (P<0.0001) and ˜30% compared to LysM-Cre/Nrp1^(+/+) mice (P=0.0008)) (FIGS. 8C, D). In turn, significant reductions in destructive pre-retinal neovascularization associated with ischemic retinopathy were observed (˜36% when compared to wild-type (P=0.0008) and ˜34% compared to LysM-Cre/Nrp1+I+ mice (P=0.0013)) (FIGS. 8E, F).

Example 10 Preparation of Soluble SEMA3A Neutralizing Traps

High affinity traps to inhibit/neutralize SEMA3A were generated. These traps were derived from Neuropilin 1 (NRP1) and were optionally coupled to 6X-His tag or FC proteins (see FIGS. 19, 20 and 27, and Table 1). Various variants comprising either the entire NRP1 extracellular domain or functional variants capable of maintaining SEMA3A binding were generated. Traps containing a b1 domain (which binds to VEGF) and including a neutralizing VEGF165 mutation were generated. The traps were shown to be highly expressed and secreted in transformed human cells. Simple purification and formulation protocols were developed to produce trap samples for SAR and in vivo efficacies studies to follow.

Methods

Cell culture and material. The human Neuropilin 1 (GenBank™ accession NM_003873, SEQ ID NO: 66) was acquired from Origene Inc. The Origen clone comprises a conservative mutation at amino acid 140 which changes the leucine for an isoleucine. The 293T (ATCC) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The pFUSE-hIgG1-Fc1 vector was purchased from InvivoGen Inc.

Cloning. The extracellular domain of Neuropilin-1 (residues 1-856), or portions of it, were PCR amplified from Origene clone RC217035 using the Phusion™ high fidelity polymerase (New England Biolabs) and cloned in the EcoR1-BgIII of pFUSE-hIgG1-Fc1 in frame with the human FC-1 coding sequence. Constructs coding for the soluble versions of the traps were generated by inserting a sequence coding for a TEV protease cleavage site followed by 6X His residues and a stop codon upstream of the FC coding portion of the corresponding FC constructs. Additional deletions (b1, b1b2) or VEGF165 binding mutations (e.g., Y297A) were introduced using the Q5 site directed mutagenesis kit (NEB). All constructs sequences were verified by Sanger sequencing (Genome Quebec). The nucleotides and amino acid sequences of the assembled traps are depicted in Appendices 1-21.

Evaluation of traps' expression in human cells. Constructs coding for the mouse and human traps were transfected in 293T cells. Cells were grown for 48 hrs post transfection in FreeStyle™ 293 medium (Invitrogen). Cell lysates were prepared from 293T cells 48 hours post-transfections. Cells were extensively washed with PBS and lysed in ice cold lysis buffer (50 mM HEPES pH7.5, 150 mM NaCL, 1.5 mM MgCl2, 1% Triton X-100 and 10% glycerol) supplemented with standard amounts of protease inhibitors (AEBSF, TPCK, TLCK, aprotinin, leupeptin, pepstatin and E64, Sigma). Cell lysates were cleared by micro centrifugation (12000 g, 20 minutes). Lysates concentrations were determined by standard micro BCA (Sigma). Equal amounts of protein were loaded on 5-20% PAGE-SDS gradient gels and transferred to PVDF (Amersham). Cleared conditioned media from transfected cells were incubated with either Protein A sepharose (Pharmacia) or Talon resin (Clontech) for FC or 6xHis tag. Resins were washed with PBS and diluted in 2×PAGE-SDS sample buffer prior to gel separation and transfer. The antibody used in immunoblottings were the anti-human Neuropilin-1 (Cell signaling), the mouse monoclonal anti-6X-HIS (In Vitrogen) and the reporter HRP linked anti-human, mouse and rabbit IgG (BioRAD). All antibodies were used at a 1/2000 dilution. Chemiluminescent signal was captured using a Fuji imaging system after incubation of membranes with ECL (Amersham).

Traps expression and purification. 293-T cells were transfected with plasmids encoding the various traps by either the Polyethylamine (PEI) or the calcium phosphate precipitation standard transfections methods. The next day cells were washed twice with serum free media and fed with serum free complete media (Free style 293 media, InVitrogen). Conditioned medium were collected after 60-72 hrs of growth in serum free media and cleared from cellular debris by swing bucket centrifugation (2000 RPM, 20 minutes). FC traps were purified from conditioned media of transfected 293T cells by passage on Protein A or G sepharose (Pharmacia) followed by extensive washes with PBS and elutions with 0.1 M glycine pH 3.0. Elution fractions were neutralised immediately by the addition of 1/10 volume 1 M Tris pH 8 and 1/10 volume of 10×PBS pH 7.4. Soluble 6X HIS tagged traps were purified from conditioned media of transfected 293T cell by passage on Talon agarose (Clontech) followed by extensive washes with PBS and stepwise imidazole elutions (Range 10-150 uM typically). Samples of purification fractions of traps were analysed on 5-15% or 5-20% gradient PAGE-SDS gels. Gel were stained using the Safely Blue staining kit (InVitrogen).

Sterile formulation of purified traps for in vivo injections. Purifications elution fractions from 40 ml of conditioned media were pooled and diluted to a total volume of 10 ml in PBS. Diluted trap proteins were sterilized by filtration through a 0.2 uM low protein binding filter (Progene). Protein solutions were concentrated and buffer exchanged with PBS on sterile PES concentration devices (Pierce, nominal MWCO 30 KD). Sterile concentrated Traps samples (˜30-50 ul) were analysed and stained on PAGE-SDS as described above.

Example 11 Affinity of Traps for SEQMA3A and VEGF

Production of AP-VEGF₁₆₅. the coding sequence of the human VEGF165 variant 1 (NM_001025366) was sub-cloned in the pAPtag5 vector (GenHunter), in-frame with an Alkaline Phosphatase domain (AP-VEGF165). HEK293T cells were transfected with the AP-VEGF165 construct using a polyethylenimine (PEI) transfection method. Following the overnight transfection step, cells were cultured for an additional 60 hr in serum free media (In vitrogen). The cell media were collected and concentrated on a PES device (Pierce). The concentrated AP-VEGF165 ligand was analysed on PAGE-SDS and quantified using SimplyBlue safe stain (Life technologies).

Sema 3A and AP-VEGF₁₆₅ binding assays. Saturation curves for the determinations of KD of binding to SEMA 3A or VEGF165 were obtained as follow. Wells of high protein binding 96 well plates (Maxisorp, Nunc) were coated with purified traps diluted in PBS and blocked afterward with binding buffer (PBS containing 2% casein and 0.05% Tween 20). The SEMA3A-FC (R&D systems) or AP-VEGF165 ligands were diluted in binding buffer over an extensive range of concentrations and added to wells. Following an overnight incubation, wells were washed with PBS containing 0.05% tween. Bound SEMA3a-FC was detected using an HRP-linked anti-Human IgG (Biorad) and ECL substrate (Pierce). Alternatively, bound AP-VEGF165 was detected using CPD star substrate (Roche). The Chemiluminescent signal was acquired on a TECAN reader. Dissociation constant (KD) were determined by non-linear curve fitting using the Graph Pad prism software.

The relative affinity of traps of the present invention to SEMA3A and VEGF has been assessed. Traps were prepared as described in Example 10. Schematic representation of traps tested is also provided in FIG. 19.

TABLE 4 Dissociation constant of SEMA3A and VEGF for various Traps SEMA 3A-FC VEGF165 SEQ ID NOs: Trap binding (nM) binding (nM) (aa and nts) G 0.8  6.75 SEQ ID NOs: 38, 39 O 1.05 N.D. SEQ ID NOs: 40, 41 M 0.95 20.13 SEQ ID NOs: 42, 43 N >1000 >250      SEQ ID Nos: 44, 45 R 6.15 N.D. SEQ ID NOs: 46, 47 W 1.14 20.73 SEQ ID NOs: 52, 53 Y >750 N.D. SEQ ID NOs: 56, 57 Z 4.44 66.96 SEQ ID NOs: 62, 63 AB N.D. 29.51 SEQ ID NOs: 58, 59 AC 4 No binding SEQ ID NOs: 60, 61

Soluble NRP1 traps of the present invention bind more efficiently to SEMA3A than VEGF. Such preference for SEMA3A was found surprising since SEMA3A and VEGF are considered to normally have the same general affinity for NRP1. Increased affinity for SEMA3A may be advantageous in conditions where SEMA3A inhibition is preferred over inhibition of VEGF and may reduce side effects associated with VEGF inhibition.

Example 12 Therapeutic Intravitreal Administration of Soluble NRP1 Reduces MP Infiltration and Pathological Neovascularization in Retinopathy

To determine the translational potential of the above findings, a soluble recombinant mouse (rm)NRP1 mTrap 1 polypeptide (FIGS. 19C and 20R comprising domains a1, a2, b1, b2 and c of SEQ ID NO.25) was next employed as a trap to sequester OIR-induced ligands of NRP1. A single intravitreal injection of rmNRP1 at P12 lead to a 30% reduction at P14 (P=0.0282) in the number of microglia present in retinas subjected to OIR (FIG. 9A). This finding attests to the potency of soluble NRP1 (1 μl of 50 μg/ml) to compromise microglial mobilization. Intravitreal administration of soluble NRP1 provoked a significant ˜40% decrease in pathological pre-retinal angiogenesis when compared to vehicle injected controls (P=0.0025) (FIGS. 9B,C). Together, these data suggest that neutralization of ligands of NRP1 is an effective strategy to reduce destructive neovascularization in retinopathy.

Example 13 Materials and Methods for Sepsis Model—Examples 14 to 19

Mouse model of sepsis. Studies were performed according to the regulations from the Canadian Guidelines for the Use of Animals in Research by the Canadian Council on Animal Care. LPS injections were delivered intra-peritoneally (i.p) in 6-8 weeks old C57BL/6 mice.

Survival assay. For generation of survival data, mice were challenged with a single intraperitoneal injection of LPS at 25 mg/kg, in a volume of nearly 100 ul adjusted to mouse weight. Mice were then monitored until reaching critical limit points defined by the Canadian Council of Animal Care.

Measurement of pro-inflammatory cytokines. For assessment of pro-inflammatory cytokines, mice were challenged i.p. with a single intraperitoneal injection of LPS at 15 mg/kg and sacrificed at various time points up to 24 hours. Tissues (Brain, Liver, Kidney) were removed and mRNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) and digested with DNase I to prevent amplification of genomic DNA. Reversed transcription was performed using M-MLV reverse transcriptase and gene expression analyzed using SybrGreen in an ABI Biosystems Real-Time PCR machine. β-actin was used as a reference gene.

Primary peritoneal macrophages culture. Adult WT or LyzMcre/NRP1fl/fl mice were anesthetized with 2% isoflurane in oxygen 2 L/min and then euthanized by cervical dislocation. Then, a small incision in abdominal skin of mouse was performed. Skin was pulled to each size of the mouse and peritoneal cavity was washed with 5 ml of PBS plus 3% FBS for 2 min. Then, the harvested cells were centrifuged for 5 min at 1000 rpm, resuspended in medium (DMEM F12 plus 10% FBS and 1% Streptomycin/Penicillin) and plated. After 1 h of culture at 37° C. under a 5% CO₂ atmosphere the medium was changed.

Cytometric Bead Array (CBA). CBA was performed according to manufacturer's guidelines (BD Bioscience). Macrophages were isolated from wild type or LyzMcre/NRP1fl/fl mice and subjected to SEMA3A (100 ng/ml) or vehicle for 12 hours and processed by CBA.

Trap and anti-VEGF antibody administration. Mice experimental model of sepsis were treated with human or mice NRP1 trap-1 (FIGS. 19B, C and 20A, 20R, SEQ ID NO: 25 or SEQ ID NO: 83) or VEGF neutralizing antibody (R&D Systems, AF-493-NA).

Experimental design: 3 mice per group. Groups: 1—Vehicle, 2—LPS, and 3—LPS+NRP1 Trap 1-Vehicle: NaCl, 2-LPS: 15 mg/kg; and 3-LPS+NRP1-trap: Mice received i.v. a single injection of 4 ug (in a volume of 100 uL) of recombinant mouse NRP1-trap corresponding to 0.2 mg/kg, few minutes after LPS injection.

Permeability tests. For permeability assays, mice were challenged i.p. with a single intraperitoneal injection of LPS at 15 mg/kg, and sacrificed 24 hrs later for tissue sampling. Changes in liver, kidney, and brain vascular permeability were assessed by quantifying Evans Blue (EB) extravasation in tissue. After 24 hrs, a solution of 10 mg/ml of EB was injected intravenously (55 mg/kg). Two hours later, mice were sacrificed and perfused through the heart with PBS. Tissues were then removed, allowed to dry at room temperature 24 hrs, and dry weights were determined. EB was extracted in formamide overnight at 65° C. EB was then measured at 620 and 740 nm in spectrophotometer.

Real-time PCR analysis. RNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) and digested with DNase I to prevent amplification of genomic DNA. Reversed transcription was performed using M-MLV reverse transcriptase (Life Technologies) and gene expression analyzed using SybrGreen (BioRad) in an ABI Biosystems Real-Time PCR machine. β-actin was used as a reference gene. See Table 3 below for details on the sequence of the oligonucleotides used.

TABLE 3 Primer sequences used for RT-PCR analysis SEQ ID Target Primer sequence NO: β-actin  GAC GGC CAG GTC ATC ACT ATT G SEQ ID  (fwd) NO: 85 β-actin  CCA CAG GAT TCC ATA CCC AAG A SEQ ID  (rev) NO: 86 SEMA3A  GCT CCT GCT CCG TAG CCT GC SEQ ID  (fwd) NO: 87 SEMA3A  TCG GCG TTG CTT TCG GTC CC SEQ ID  (rev) NO: 88 VEGF  GCC CTG AGT CAA GAG GAC AG SEQ ID  (fwd) NO: 89 VEGF  CTC CTA GGC CCC TCA GAA GT SEQ ID  (rev) NO: 90 Tnf-α CCC TCA CAC TCA GAT CAT CTT CT SEQ ID  (fwd) NO: 91 Tnf-α GCT ACG ACG TGG GCT ACA G SEQ ID  (rev) NO: 92 IL-1β CTG GTA CAT CAG GAC CTC ACA SEQ ID  (fwd) NO: 93 IL-1β GAG CTC CTT AAC ATG CCC TG SEQ ID  (rev) NO: 94 IL-6  AGA CAA AGC CAG AGT CCT TCA GAG A SEQ ID  (fwd) NO: 095 IL-6  GCC ACT CCT TCT GTG ACT CGA GC SEQ ID  (Rev) NO: 96

Example 14 Semaphorin 3A is Upregulated in Several Organs During Septic Shock

Given the link between SEMA3A, NRP1 and the innate immune response in OIR (as demonstrated in Examples 2-9 above), the implication of the NRP1-dependent cellular response in general systemic inflammation was next assessed. This was first explored by determining the kinetics of SEMA3A expression during septic shock.

LPS was administrated (15 mg/kg) to 6-8 weeks old C57BL/6 mice (n=5) and mice were sacrificed at 0, 4, 8, 12 and 24 hours following LPS administration. Key organs such as brain, kidney, lung and liver were collected and mRNA isolated. Levels of SEMA3A mRNA were robustly induced in all organs analyzed as soon as 6 hours after LPS injection and persisted for 24 hours (FIG. 11 A-D). Similarly, expression levels of another NRP1 ligand, VEGF, were also profoundly increased in kidney (FIG. 11B), lung (FIG. 11C) and liver (FIG. 11D) within the first 6 hours of septic shock. Increases in classical pro-inflammatory cytokines TNF-α and IL1-β rose at 6 hours post LPS administration and diminished similarly to VEGF mRNA (FIG. 12). Hence, of all investigated mediators of inflammation, SEMA3A had a long-term kinetic profile and stayed elevated for at least 24 hours following induction of sepsis. This particular expression profile for SEMA3A suggests that its contribution to septic shock may be long lasting when compared to other cytokines.

Example 15 SEMA3A Induces Secretion of Pro-Inflammatory Cytokines in Myeloid Cells Via NRP1

Given the contribution of monocytes and myeloid cells to the acute inflammatory response and the presence of NRP1 on myeloid cells, the contribution of SEMA3A and myeloid-resident NRP1 in the production of inflammatory cytokines was determined.

Isolated macrophages were exposed to SEMA3A (100 ng/ml) or vehicle and the production of cytokines was analyzed by Cytometric Bead Array (CBA). Results presented in FIG. 13 indicate that SEMA3A can induce the production/secretion of pro-inflammatory cytokines, known to contribute to septic shock such as IL-6 (FIG. 13A) and TNF-α (FIG. 13B). Of particular importance, a specific knockout of NRP1 (LyzM/NRP1^(fl/fl)) in myeloid cells abrogated SEMA3A-induced production of IL-6 and TNF-α. Notably, vehicle-treated control LyzM/NRP1^(fl/fl) macrophages showed lesser production of IL-6, TNF-α and IL-1β then wild-type controls, highlighting the role of myeloid-resident NRP1 in sepsis-induced inflammation.

Example 16 Deficiency in Myeloid-Resident NRP1 Reduces Production of Pro-Inflammatory Cytokines In Vivo in Sepsis

Because myeloid-resident NRP1 was important for the release of pro-inflammatory cytokines such as IL-6 and TNF-α in vitro, its contribution was next explored in vivo. LyzM/NRP1^(fl/fl) and control wild-type mice were administered vehicle or LPS (15 mg/kg) and brains and livers were collected 6 hours post LPS injection. Real-time PCR analysis of TNF-α (FIGS. 14A,C) and IL-1b (FIG. 14B,D) levels revealed a robust drop in these cytokines in LyzM/NRP1^(fl/fl). These results underscore the profound contribution of NRP1 and its ligands to the development of sepsis in vivo.

Example 17 Inhibition of NRP1 Signalling Prevents Sepsis-Induced Barrier Function Breakdown

One of the pathological features of severe septic shock though to contribute to organ failure is the compromise of blood barriers (blood and air in lung, blood and urine in the kidney, blood and bile in liver, and humoral molecules in the brain). Given a role for SEMA3A in the breakdown of the blood retinal barrier (46) and the present novel data on the expression of SEMA3A during sepsis, the effect of neutralizing SEMA3A with a trap derived from the extracellular domain of human NRP1 was assessed (Trap-1, without FC, FIG. 19B, SEQ ID NO:83). Using an Evans Blue Permeation (EBP) assay, we found that in all organs studied namely brain (FIG. 15A), kidney (FIG. 15B) and liver (FIG. 15C), a pronounced reduction in LPS-induced barrier function breakdown was observed when mice were treated with 4 ug of NRP1 derived trap (0.2 mg/kg, i.v.). These results strongly suggest that traps of soluble NRP1 and their derivatives are compelling therapeutic agents to counter sepsis.

Example 18 NRP1-Derived Trap Protects Against Sepsis

To determine the therapeutic benefits of neutralization of NRP1 ligands or NRP1 inhibition during sepsis, survival studies were performed. A high dose of LPS (25 mg/kg) was administered to mice. Mice were then monitored, and ethically sacrificed, when appropriate endpoints were achieved. In the second group, mice were injected i.v. with 4 ug of recombinant Trap-1 without FC (0.2 mg/kg, FIGS. 19B and 20A, SEQ ID NO: 83) followed by LPS intraperitoneal injection. In the control group, 5/5 mice (100%) died within first 30 hrs (FIG. 16A) following LPS injection. Conversely, all mice treated with the trap were still alive after 30 hours and showed significant improved survival rate after 60 hours (3/5). Mortality was thus reduced from 100% (in the control group) after 30 hours to 40% (FIG. 16A) after 60 hours. Furthermore, 40% of Trap treated-mice remained alive 80 hours following LPS injection. Thus, survival time was at least doubled in 60% of the case and almost tripled in 40% of the case when cell signaling through NRP1 was inhibited.

Similar results were obtained with mice harboring a specific knock out of NPR1 in myeloid cells (FIG. 16B). Absence of NRP1 in myeloid cells increased survival time and reduced sepsis-induced mortality (3/5) from 100% to 40% (FIG. 16B) after 30 hours and from 100% to 40% after 60 hours. Also, 40% of NRP1 K.O. mice remained alive 80 hours following LPS injection.

Taken together, these results highlight the therapeutic value of inhibiting NRP1-dependent cell signaling in sepsis treatment.

Example 19 NRP1-Derived Trap Lowers Production of Inflammatory Cytokines in Septic Shock

Given the therapeutic benefit of NRP1-trap on survival rates in septic shock, the impact of neutralization of NRP1 ligands on production of inflammatory cytokines during septic shock was next determined. Wild-type mice were administered i) vehicle (n=3); ii) LPS (15 mg/kg) (n=3) or iii) LPS and NRP1 mouse Trap 1 (without FC, FIG. 19C SEQ ID NO: 25, but without FC region) and brains were collected 6 hours post LPS injection. Injection of NRP1 trap-1 profoundly reduced production of TNF-α (FIG. 17A) and IL-6 (FIG. 17B). Similarly, mice with NRP1 deficient myeloid cells (LyzM-Cre/Nrp^(fl/fl)) (n=3) produced considerably less TNF-α and IL-6, underscoring the contribution of this cellular pathway to the progression of septic shock.

Example 20 Materials and Method for the Cerebral Ischemiaistroke Model Described in Example 21

The mice used in this study were 2- to 3-month old male C57Bl/6 mice (22-28 g).

MCAO model. MCAO mouse model was performed using the intraluminal suture technique described by Rousselet et al. (66). Briefly, mice were anesthetized in a chamber with 3% isoflurane in oxygen (1 L/min) and analgesized with buprenorphine (0.1 mg/kg body weight subcutaneously). Anesthesia was maintained during the operation using 1.5% isoflurane in oxygen provided via a face mask. The rectal temperature was recorded and kept stable at 37±0.5° C. with a heating pad. After a midline incision at the neck, the right carotid bifurcation was exposed and the common carotid artery (CCA) was temporarily occluded using 5-0 silk suture. The bifurcation of the right internal common carotid artery (ICA) and external common carotid artery (ECA) was separated. A permanent suture was placed around the ECA, as distally as possible, and another temporary suture slightly tight was placed on the ECA distal to the bifurcation. The right ICA was temporarily occluded with 5-0 silk suture to avoid bleeding. Then, a small hole in the ECA was cut between permanent and temporary sutures through which a 12 mm-long 6-0 silicon-coated (about 9-10 mm was coated with silicon) monofilament suture was introduced. The filament was advanced from the ECA into the lumen of the ICA until it blocked the origin of the middle cerebral artery (MCA) in the circle of Willis. Sham animals were obtained by inserting the monofilament into the CCA, but without advancing it to the MCA. The suture on the ECA was tightly tied to fix the monofilament in position. Thirty minutes after MCAO, the monofilament was completely removed to allow reperfusion. The temporary suture on the CCA was also removed to allow blood recirculation. After the wound was closed, 1 ml of saline solution was injected subcutaneously to avoid postsurgical dehydration. The mouse was placed in a cage and kept on the heating pad for 1 h. Meantime, when the mouse was fully awake from anesthesia, it was checked for some basic motor deficits (circling while walking and bending while hold by tail; indicators of the success of the operation) and NRP1— Trap-1 without FC (FIG. 19B SEQ ID NO:83) at the dose of 0.4 ug in 125 ul of PBS was administered to the tail vein (about 15 min after reperfusion had been started). Control animals operated in the same way as NRP1-treated animals received, after MCAO, a vehicle (PBS). Because post-surgical weight loss is generally observed, mashed food was placed in a Petri dish to encourage eating.

Determination of infarct volume. Following neurological evaluation (see section below) performed 24 h after MCAO the animals were deeply anesthetized with 3% isoflurane in oxygen (1 L/min) and decapitated. The brains were immediately isolated and transferred into isopentane cooled on dry ice and then stored at −80° C. Then, the frozen brains were coronally cut into 20-μm sections in a cryostat at −22° C. and every 15th slice was mounted on positively charged glass slides. Cerebral sections were stained with cresyl violet for 15 min. Each section was photographed. The areas of infarction were delineated on the basis of the relative lack of staining in the ischemic region and measured by using NIH ImageJ software. Infarct area in each section was determined as the total area of the contralateral hemisphere minus the non-affected area of the ipsilateral hemisphere.

Neurological evaluation. One hour after operation, as well as 24 h after MCAO, animals were subjected to a series of motor tests performed. The examinations and scoring were as follows: 0, Normal; 1, Contralateral front or rear limb flexion upon lifting of the whole animal by the tail; 2, Circling to the contralateral side while walking and C-shaped lateral bending while hold by tail; 4 Circling to the contralateral side while walking and C-shaped lateral bending while hold by tail with limb flexion; 5, Comatose or moribund. The magnitude of the obtained neuroscore is directly proportional to the severity of impairment.

Example 21 NRP1-Trap Protects Against Cerebral Ischemia and Stroke

In order to assess the outcome of SEMA3A neutralization on cerebral ischemia or stroke, adult (8-12 week-old) mice were subjected to the transient middle cerebral artery occlusion (MCAO) model. Experimental details are provided in Example 20. Briefly, following termination of MCAO, mouse NRP1-trap (Trap-1, without FC), 0.4 ug in 125 ul of PBS, (FIG. 19C, FIG. 20R SEQ ID NO: 25) was administered to the tail vein (about 15 min after reperfusion had been started). In order to visualize brain damage induced by MCAO, coronal cerebral sections were stained with cresyl violet. On each section, the unstained area corresponded to the ischemic region of the brain (FIG. 18A). Measurement of these areas on serial coronal sections revealed that 24 h after MCAO, the infarcted zone constituted 48% of the ipsilateral hemisphere in occluded mice compared to sham operated animals whose brains were not injured. NRP1 treatment reduced brain damage; the infarct volume of the ipsilateral hemisphere was decreased by 80% (FIG. 18B,C).

Neurological impairment was assessed by neurological scoring of the presence of limb flexion, C-shaped lateral bending of the body and circling movements. MCAO mice that were not showing circling and bending behaviour 1 hour after operation were excluded from the further study (FIG. 18D). Forelimb or hindlimb flexion, C-shaped lateral bending of the body, circling movements were observed in mice subjected to MCAO compared to sham operated animals. NRP1 treatment dramatically improved neurological scores of ischemic mice by 60% compared to non-treated MCAO mice when evaluated 24 hours after surgery (FIG. 18E).

Taken together, these results show that inhibition of the NRP1 pathway protects against cerebral ischemia and stroke and reduce the neurological impairment associated with cerebral ischemia and stroke.

Example 22 Neuropilin-Derived Traps Enhance Vascular Regeneration and Prevent Pathological Neovascularization in Ischemic Retinas in Mouse Model of Diabetic Retinopathy and Retinopathy of Prematurity

Pathological vascular degeneration as well as pre-retinal vascular proliferation were studied using the well-established mouse model of oxygen-induced proliferative retinopathy (OIR)(Smith et al., 1994). This model is based on retinopathy of prematurity (ROP) and is regularly used as a proxy for the proliferative (angiogenic phase) of diabetic retinopathy and ROP.

Nursing mothers and their pups were exposed to 75% oxygen from P7-P12. Both vaso-degenerative (assessed at P12) and vaso-proliferative (assessed at P17) phases are present and are highly reproducible making evaluation of interventions on disease progression accurate and swift. Trap G (SEQ ID NO: 38), or Trap M (lacking the b2 and c domains, SEQ ID NO: 42), was injected into the vitreous at P12 (1 ul at 0.5 ug/ul). Dissected retinas were flatmounted and incubated overnight with fluoresceinated isolectin B4 (1:100) in 1 mM CaCl₂ to determine extent of avascular area or neovascularization area at P17. Avascular areas were determined in lectin stained retinas as zones devoid of staining. Neovascularization was determined as areas of saturated lectin staining which demarcates pre-retinal tufts (54, 55).

Trap G, was shown to effectively enhance vascular regeneration by over 40% when compared to vehicle control (FIG. 23B). Similarly, Trap G was shown to inhibit pathological neovascularization by ˜45% (FIG. 23C). Trap-M enhanced vascular regeneration by ˜60% (FIG. 23B) and inhibited pathological neovascularization by ˜60% when compared to vehicle controls (FIG. 23C). Hence, Trap M, with compromised VEGF binding, more effectively prevents pathological angiogenesis and more readily leads to enhanced vascular regeneration in the ischemic retina.

Example 23 Neuropilin-Derived Traps Decrease Vascular Leakage in Diabetic Retinas

The influence of Traps on vascular leakage/permeability in diabetic retinopathy was also studied in the streptozotocin (STZ) model of Type 1 diabetes. STZ (55 mg/kg) was administered over 5 consecutive days to ˜6 week-old C57BL/6J mice and glycemia was monitored. Mice were considered diabetic if their non-fasted glycemia was higher than 17 mM (300 mg/dL). Mice were administered intravitreally with 0.5 ug (0.5 ug/ul) of Trap G (SEQ ID NO: 38) or M (SEQ ID NO: 42) or with mouse anti-VEGF antibody (AF-493-NA, from R&D) at 6 and 7 weeks after STZ administration. Alternatively, mice were injected intravitreally at 12 and 13 weeks post STZ and vascular permeability assessed at 14 weeks. Mice were hyperglycemic/diabetic at least 3 weeks prior to intravitreal injections with SEMA traps (see FIG. 24A) or anti VEGF antibody. Retinal vascular leakage was determined by Evans Blue assay at 8 weeks post STZ injections as follows. Retinal Evans Blue (EB) permeation was performed using 3 retinas per reading. Evan Blue was injected at 45 mg/kg intravenously and allowed to circulate for 2 hours prior to retinal extraction. Evans Blue Permeation was quantified in retinas by fluorimetry (620 nm max absorbance−740 nm min absorbance (background) with a TECAN Infinite® M1000 PRO. Evan Blue Permeation (EBP) [measured in uL/(grams*hour)] was calculated as follows: [EB (ug)/Wet retinal weight (g)]/[plasma EB (ug/uL)*Circulation time (hours)]. Evans Blue permeation was expressed relative to controls.

Both Trap-G (SEQ ID NO: 38) and Trap-M (SEQ ID NO: 42) significantly reduced vascular permeability by over 40% (FIG. 24B). The mouse anti-VEGF antibody (AF-493-NA) did not prevent vascular permeability at this early stage. Trap G was effective at reducing vascular leakage as was the anti-VEGF neutralizing Ab at P17 (FIG. 24C), *p<0.05, n=4, from 12 animals.

Example 24 Neuropilin-Derived Traps Decrease Choroidal Neovascularization in Model of Age-Related Macular Degeneration

The effect of NRP1 trap G (SEQ ID NO: 38) on choroidal neovascularization (CNV) was determined in a mouse model of age-related macular degeneration (AMD). To induce CNV and thus mimic wet AMD in mice, laser coagulations on 6-8 week old mice (1-2 disc diameters) were performed from the papillae using an Argon laser (532 nm) mounted on a Coherent slit lamp (400 mW, 50 ms and 50 μm) (Combadiere et al., 2007). Following laser burn, treated mice were injected intravitreally with 0.5 ug of Trap G. Fourteen days (P14) later, choroids were radially incised, flat-mounted and stained with the endothelial cell marker fluoresceinated Isolectin B4 (animals were also optionally perfused with fluorescein dextran to visualize luminal vessels) and volumes of CNV were measured by scanning laser confocal microscopy (Takeda et al., 2009).

Trap G was shown to significantly reduce choroidal neovascularization at day 14 post laser-burn (FIG. 25 B).

The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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1-69. (canceled)
 70. A method of treating or preventing (i) neurological damage induced by a stroke or cerebral ischemia; (ii) septic shock; or (iii) inflammation, comprising inhibiting NRP1-dependent cell-signaling in a subject.
 71. The method of claim 70, for treating or preventing (i) neurological damage induced by a stroke or cerebral ischemia; or (ii) septic shock, comprising administering (a) a NRP1 trap comprising a NRP1 polypeptide or a functional fragment or variant thereof; (b) an anti-NRP1 antibody; or (c) a NRP1 shRNA or siRNA, to the subject.
 72. The method of claim 71, wherein said method comprises administering a NRP1 trap comprising a NRP1 polypeptide or a functional fragment or variant thereof to the subject.
 73. The method of claim 71, wherein said method reduces secretion of IL-6, IL-1β, TNFα and/or recruitment of mononuclear phagocytes (MPs) at a site of injury in the subject.
 74. The method of claim 72, wherein said NRP1 polypeptide or functional fragment or variant thereof corresponds to (i) NRP1 isoform 2; (ii) the extracellular domain of NRP1 isoform 1; or (iii) a functional fragment or variant of (i) or (ii).
 75. The method of claim 74, wherein said NRP1 trap comprises a NRP1 polypeptide consisting of (i) amino acids 22 to 609 of a NRP1 polypeptide as set forth in SEQ ID NO: 65; (ii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 66; (iii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 69; or (iv) a functional fragment or functional variant of (i), (ii) or (iii).
 76. The method of claim 74, wherein said NRP1 trap comprises a NRP1 polypeptide: (a) consisting of domains a1, a2, b1, b2, and c and of said NRP1 polypeptide; (b) consisting of domains a1, a2, b1 and b2 of said NRP1 polypeptide; (c) consisting g of domains a1, a2, and b1 of said NRP1 polypeptide; (d) consisting of domains a1 and a2 of said NRP1 polypeptide; (e) comprising domain b1 of said NRP1 polypeptide, wherein said domain b1 comprises at least one point mutation at an amino acid residue corresponding to (i) tyrosine 297; (ii) aspartic acid 320 and/or (iii) glutamic acid 319, of said NRP1 polypeptide, wherein said at least one mutation reduces or abrogates binding to VEGF₁₆₅; (f) lacking completely or partially domain c of said NRP1 polypeptide; (g) lacking completely or partially domain b1 of said NRP1 polypeptide; (h) lacking completely or partially domain b2 of said NRP1 polypeptide; (i) lacking domains b1 and b2 of said NRP1 polypeptide; or (j) a functional fragment or variant of any one of (a) to (i).
 77. The method of claim 74, wherein said NRP1 trap comprises a NRP1 polypeptide consisting of: (i) SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113 lacking a signal peptide or a functional variant thereof; or (ii) SEQ ID NO: 1, 3, 5, 7, 9, 12, 13, 15, 17, 19, 21, 23, 31, 33, 35, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 83 or a functional variant thereof.
 78. The method of claim 77, wherein said NRP1 trap comprises a NRP1 polypeptide consisting of: (i) SEQ ID NO: 9, 40 or 42; (ii) SEQ ID NO: 105 or 107 without a signal peptide; or (iii) a functional variant of (i) or (ii).
 79. A NRP1 trap comprising a NRP1 polypeptide consisting of: (i) SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113 lacking a signal peptide or a functional variant thereof; or (ii) SEQ ID NO: 1, 3, 5, 7, 9, 12, 13, 15, 17, 19, 21, 23, 31, 33, 35, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 83 or a functional variant thereof.
 80. The NRP1 trap of claim 79, wherein said NRP1 polypeptide consists of: (i) SEQ ID NO: 9, 40 or 42; or (ii) SEQ ID NO: 105 or 107 without a signal peptide; or (iii) a functional variant of (i) or (ii).
 81. A NRP1 trap comprising a NRP1 polypeptide consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113 or a functional variant thereof and further comprising a protein purification domain.
 82. The NRP1 trap of claim 81, further comprising a protease or peptidase cleavage site enabling said protein purification domain to be removed from said NRP1 trap.
 83. The NRP1 trap of claim 81, wherein said protein purification domain is a histidine tag or a FC fragment.
 84. The NRP1 trap of claim 82, wherein said protease or peptidase cleavage site is a TEV protease cleavage site.
 85. A nucleic acid encoding the NRP1 trap of claim
 79. 86. An expression vector comprising the nucleic acid of claim
 85. 87. A host cell comprising the vector of claim
 86. 88. A composition comprising the NRP1 trap of claim 79 and a suitable pharmaceutical carrier.
 89. The method of claim 77, wherein said functional variant is (a) an allelic variant or (b) a variant comprising at least one conservative amino acid substitution, wherein the at least one conservative amino acid substitution is located at a position corresponding to a variable amino acid in the NRP1 polypeptide sequence set forth in SEQ ID NO:
 69. 90. The NRP1 trap of claim 79, wherein said functional variant is (a) an allelic variant or (b) a variant comprising at least one conservative amino acid substitution, wherein the at least one conservative amino acid substitution is located at a position corresponding to a variable amino acid in the NRP1 polypeptide sequence set forth in SEQ ID NO:
 69. 